Mammalian receptor proteins; related reagents and methods

ABSTRACT

Nucleic acids encoding mammalian, e.g., primate, receptors, purified receptor proteins and fragments thereof. Antibodies, both polyclonal and monoclonal, are also provided. Methods of using the compositions for both diagnostic and therapeutic utilities are described.

[0001] This filing claims benefit of U.S. Provisional Patent ApplicationNo. 60/203,426, filed May 10, 2000 which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods foraffecting mammalian physiology, including immune system function. Inparticular, it provides methods to regulate development and/or theimmune system. Diagnostic and therapeutic uses of these materials arealso disclosed.

BACKGROUND OF THE INVENTION

[0003] Recombinant DNA technology refers generally to techniques ofintegrating genetic information from a donor source into vectors forsubsequent processing, such as through introduction into a host, wherebythe transferred genetic information is copied and/or expressed in thenew environment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host. See, e.g.,Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, (2d ed.)vols. 1-3, CSH Press, N.Y.

[0004] For some time, it has been known that the mammalian immuneresponse is based on a series of complex cellular interactions, calledthe “immune network”. Recent research has provided new insights into theinner workings of this network. While it remains clear that much of theimmune response does, in fact, revolve around the network-likeinteractions of lymphocytes, macrophages, granulocytes, and other cells,immunologists now generally hold the opinion that soluble proteins,known as lymphokines, cytokines, or monokines, play critical roles incontrolling these cellular interactions. Thus, there is considerableinterest in the isolation, characterization, and mechanisms of action ofcell modulatory factors, an understanding of which will lead tosignificant advancements in the diagnosis and therapy of numerousmedical abnormalities, e.g., immune system disorders.

[0005] Lymphokines apparently mediate cellular activities in a varietyof ways. See, e.g., Paul (ed. 1996) Fundamental Immunology 3d ed., RavenPress, New York; and Thomson (ed. 1994) The Cytokine Handbook 2d ed.,Academic Press, San Diego. They have been shown to support theproliferation, growth, and/or differentiation of pluripotentialhematopoietic stem cells into vast numbers of progenitors comprisingdiverse cellular lineages which make up a complex immune system. Properand balanced interactions between the cellular components are necessaryfor a healthy immune response. The different cellular lineages oftenrespond in a different manner when lymphokines are administered inconjunction with other agents.

[0006] Cell lineages especially important to the immune response includetwo classes of lymphocytes: B-cells, which can produce and secreteimmunoglobulins (proteins with the capability of recognizing and bindingto foreign matter to effect its removal), and T-cells of various subsetsthat secrete lymphokines and induce or suppress the B-cells and variousother cells (including other T-cells) making up the immune network.These lymphocytes interact with many other cell types.

[0007] Research to better understand and treat various immune disordershas been hampered by the general inability to maintain cells of theimmune system in vitro. Immunologists have discovered that culturingmany of these cells can be accomplished through the use of T-cell andother cell supernatants, which contain various growth factors, includingmany of the lymphokines.

[0008] Various growth and regulatory factors exist which modulatemorphogenetic development. Many receptors for cytokines are known.Often, there are at least two critical subunits in the functionalreceptor. See, e.g., Heinrich, et al. (1998) Biochem. J. 334:297-314;Gonda and D'Andrea (1997) Blood 89:355-369; Presky, et al. (1996) Proc.Nat'l Acad. Sci. USA 93:14002-14007; Drachman and Kaushansky (1995)Curr. Opin. Hematol. 2:22-28; Theze (1994) Eur. Cytokine Netw.5:353-368; and Lemmon and Schlessinger (1994) Trends Biochem. Sci.19:459-463.

[0009] From the foregoing, it is evident that the discovery anddevelopment of new soluble proteins and their receptors, including onessimilar to lymphokines, should contribute to new therapies for a widerange of degenerative or abnormal conditions which directly orindirectly involve development, differentiation, or function, e.g., ofthe immune system and/or hematopoietic cells. In particular, thediscovery and understanding of novel receptors for lymphokine-likemolecules which enhance or potentiate the beneficial activities of otherlymphokines would be highly advantageous. The present invention providesnew receptors for ligands exhibiting similarity to cytokine likecompositions and related compounds, and methods for their use.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to novel receptors related tocytokine receptors, e.g., primate, cytokine receptor-like molecularstructures, designated DNAX Cytokine Receptor Subunits (DCRS), and theirbiological activities. In particular, it provides description of onesubunit, designated DCRS5. It includes nucleic acids coding for thepolypeptides themselves and methods for their production and use. Thenucleic acids of the invention are characterized, in part, by theirhomology to cloned complementary DNA (cDNA) sequences enclosed herein.Additionally, the invention provides matching of the p40/IL-B30 ligandwith receptor subunits DCRS5 and IL-12Rβ1, which pairing providesinsight into indications for use of the agonists and antagonists basedupon reagents directed thereto.

[0011] The present invention provides a substantially pure orrecombinant polypeptide comprising at least ten contiguous amino acidsof the intracellular portion of SEQ ID NO:2. In certain embodiments, thepolypeptide: comprises at least 25 contiguous amino acids of theintracellular portion of SEQ ID NO:2; is recombinant, comprising theintracellular portion of SEQ ID NO:2; further comprises at least tencontiguous amino acids of the non-intracellular portion of SEQ ID NO:2;comprises at least 25 amino acids of the extracellular portion of SEQ IDNO:2; comprises the mature SEQ ID NO:2; or is a substantially purenatural polypeptide.

[0012] In others, the recombinant polypeptide: consists of the maturesequence of Table 1; is an unglycosylated polypeptide; is from a human;comprises at least 40 contiguous amino acids of SEQ ID NO:2; exhibits atleast three nonoverlapping segments of at least fifteen contiguous aminoacids of SEQ ID NO:2; is a natural polymorphic variant of SEQ ID NO:2;has a length at least about 30 amino acids; exhibits at least twonon-overlapping epitopes which are specific for a primate DCRS5; has amolecular weight of at least 30 kD with natural glycosylation; is asynthetic polypeptide; is in a sterile form; is in an aqueous orbuffered solution; is attached to a solid substrate; is conjugated toanother chemical moiety; or is physically associated with an IL-12Rβ1polypeptide.

[0013] Other embodiments of the invention provide: a substantially pureor recombinant polypeptide comprising at least two distinctnonoverlapping segments of at least six contiguous amino acids of theintracellular portion of SEQ ID NO:2; a substantially pure orrecombinant polypeptide comprising at least twelve contiguous aminoacids of the intracellular portion of SEQ ID NO:2; or a substantiallypure natural sequence polypeptide comprising mature SEQ ID NO:2. Inparticular forms, the polypeptide comprising at least two distinctnonoverlapping segments of at least six contiguous amino acids of theintracellular portion of SEQ ID NO:2 will be where: the distinctnonoverlapping segments: include one of at least twelve amino acids;include one of at least seven amino acids and a second of at least nineamino acids; include a third distinct segment of at least six aminoacids; or comprise one of R355-L373, P378-L405, V407-D426, K428-D439,P441-V452, I454-G460, I465-T587, or N592-606; or the polypeptide furthercomprises at least two distinct nonoverlapping segments of at least sixcontiguous amino acids of the extracellular portion of SEQ ID NO:2.Alternatively, the polypeptide comprising at least twelve contiguousamino acids of the intracellular portion of SEQ ID NO:2 will be onewhere: the at least twelve contiguous amino acid segment comprises oneof R355-L373, P378-L405, V407-D426, K428-D439, P441-V452, I454-G460,I465-T587, or N592-606; or the polypeptide further comprises at leasttwo distinct nonoverlapping segments of at least six contiguous aminoacids of the extracellular portion of SEQ ID NO:2. Or, the pure naturalsequence polypeptide comprising mature SEQ ID NO:2 may furthercomprising a purification or detection epitope. Such polypeptides may:consist of the mature sequence of Table 1; be an unglycosylatedpolypeptide; be from a human; comprise at least 40 contiguous aminoacids of SEQ ID NO:2; exhibit at least three nonoverlapping segments ofat least fifteen contiguous amino acids of SEQ ID NO:2; be a naturalpolymorphic variant of SEQ ID NO:2; have a length at least about 30amino acids; exhibit at least two non-overlapping epitopes which arespecific for a primate DCRS5; have a molecular weight of at least 30 kDwith natural glycosylation; be a synthetic polypeptide; be in a sterilform; be in an aqueous or buffered solution; be attached to a solidsubstrate; be conjugated to another chemical moiety; or be physicallyassociated with an IL-12Rβ1 polypeptide.

[0014] Various other compositions are provided, e.g., comprising: asubstantially pure polypeptide combined with the IL-12Rβ1 protein; orsuch a polypeptide in a carrier, wherein the carrier is: an aqueouscompound, including water, saline, and/or buffer; and/or formulated fororal, rectal, nasal, topical, or parenteral administration.

[0015] Kits are provided comprising such a polypeptide and: acompartment comprising the polypeptide; a compartment comprising anIL-12Rβ1 polypeptide; a compartment comprising a p40, IL-B30, orp40/IL-B30 polypeptide; or instructions for use or disposal of reagentsin the kit.

[0016] Antibodies and other binding compounds are provided, e.g.,comprising an antigen binding site from an antibody, which specificallybinds to the intracellular portion of the DCRS5, wherein: the bindingcompound is in a container; the polypeptide is from a human; the bindingcompound is an Fv, Fab, or Fab2 fragment; the binding compound isconjugated to another chemical moiety; or the antibody: is raisedagainst a peptide sequence of a mature polypeptide of Table 1; is raisedagainst a mature DCRS5; is raised to a purified human DCRS5; isimmunoselected; is a polyclonal antibody; binds to a denatured DCRS5;exhibits a Kd to antigen of at least 30 μm; is attached to a solidsubstrate, including a bead or plastic membrane; is in a sterilecomposition; or is detectably labeled, including a radioactive orfluorescent label. Kits are also provided comprising the bindingcompound and: a compartment comprising the binding compound; acompartment comprising: a p40 polypeptide; an IL-B30 polypeptide; aDCRS5 polypeptide; and/or an IL- 12Rβ1 polypeptide; a compartmentcomprising an antibody which binds selectively to: a p40 polypeptide; anIL-B30 polypeptide; a DCRS5 polypeptide; and/or an IL-12Rβ1 polypeptide;or instructions for use or disposal of reagents in the kit.

[0017] Also provided are methods, e.g., of producing an antigen:antibodycomplex, comprising contacting under appropriate conditions a primateDCRS5 polypeptide with an antibody, thereby allowing the complex toform. Such method may be where: the complex is purified from othercytokine receptors; the complex is purified from other antibody; thecontacting is with a sample comprising an interferon; the contactingallows quantitative detection of the antigen; the contacting is with asample comprising the antibody; or the contacting allows quantitativedetection of the antibody. Other compositions are provided, e.g.,composition comprising: a sterile binding compound, or the bindingcompound and a carrier, wherein the carrier is: an aqueous compound,including water, saline, and/or buffer; and/or formulated for oral,rectal, nasal, topical, or parenteral administration.

[0018] The invention also provides an isolated or recombinant nucleicacid encoding the DCRS5 polypeptide, wherein the: DCRS5 is from a human;or the nucleic acid: encodes an antigenic peptide sequence of Table 1;encodes a plurality of antigenic peptide sequences of Table 1; exhibitsidentity over at least thirteen nucleotides to a natural cDNA encodingthe segment; is an expression vector; further comprises an origin ofreplication; is from a natural source; comprises a detectable label;comprises synthetic nucleotide sequence; is less than 6 kb, preferablyless than 3 kb; is from a primate; comprises a natural full lengthcoding sequence; is a hybridization probe for a gene encoding the DCRS5;or is a PCR primer, PCR product, or mutagenesis primer. Cells comprisingthe recombinant nucleic acid are provided, including where the cell is:a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell;an insect cell; a mammalian cell; a mouse cell; a primate cell; or ahuman cell.

[0019] Kit embodiments include those comprising the nucleic acid and: acompartment comprising the nucleic acid; a compartment comprising anucleic acid encoding: a p40 polypeptide; an IL-B30 polypeptide; a DCRS5polypeptide; and/or an IL-12Rβ1 polypeptide; a compartment comprising: ap40 polypeptide; an IL-B30 polypeptide; a DCRS5 polypeptide; and/or anIL-12Rβ1 polypeptide; a compartment comprising an antibody whichselectively binds to: a p40 polypeptide; an IL-B30 polypeptide; a DCRS5polypeptide; and/or an IL-12Rβ1 polypeptide; or instructions for use ordisposal of reagents in the kit.

[0020] Other nucleic acid embodiments include those which: hybridizeunder wash conditions of 30 minutes at 30° C. and less than 2M salt tothe portion of SEQ ID NO:1 encoding the intracellular portion; orexhibit identity over a stretch of at least about 30 nucleotides to theintracellular portion of a primate DCRS5. Preferably, such nucleic acidwill be one wherein: the wash conditions are at 45° C. and/or 500 mMsalt; or 55° C. and/or 150 mM salt; or the stretch is at least 55 or 75nucleotides.

[0021] Therapeutic uses include methods of modulating physiology ordevelopment of a cell comprising contacting the cell with: an antagonistof p40/IL-B30 which is a complex comprising: the extracellular portionof a primate DCRS5 and/or the extracellular portion of a primateIL-12Rβ1; an antagonist of p40/IL-B30 which is an antibody which binds acomplex comprising: primate DCRS5 and/or primate IL-12Rβ1; an antagonistof p40/IL-B30 which is an antibody which binds to DCRS5; an antagonistof p40/IL-B30 which is an antibody to IL-12Rβ1; an antagonist ofp40/IL-B30 which is an antisense nucleic acid to DCRS5 or IL-12Rβ1; oran agonist of p40/IL-B30 which is an antibody which binds a complexcomprising primate DCRS5 and/or primate IL-12Rβ1. In one type of method,the contacting is with an antagonist, and the contacting is incombination with an antagonist to IL-12, IL-18, TNF, and/or IFNγ; or thecell is from a host which: exhibits signs or symptoms of a chronic TH1mediated disease; exhibits symptoms or signs of multiple sclerosis,rheumatoid arthritis, osteoarthritis, inflammatory bowel disease,diabetes, psoriasis, or sepsis; or receives an allogeneic transplant.Conversely, the method may be contacting with an agonist, and: thecontacting is in combination with IL-12, IL-18, TNF, or IFNγ; or thecell is from a host which:

[0022] exhibits signs or symptoms of a chronic Th2 response; suffersfrom a tumor, viral, or fungal growth; receives a vaccine; or suffersfrom an allergic response.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Outline

[0023] I. General

[0024] II. Activities

[0025] III. Nucleic acids

[0026] A. encoding fragments, sequence, probes

[0027] B. mutations, chimeras, fusions

[0028] C. making nucleic acids

[0029] D. vectors, cells comprising

[0030] IV. Proteins, Peptides

[0031] A. fragments, sequence, immunogens, antigens

[0032] B. muteins

[0033] C. agonists/antagonists, functional equivalents

[0034] D. making proteins

[0035] V. Making nucleic acids, proteins

[0036] A. synthetic

[0037] B. recombinant

[0038] C. natural sources

[0039] VI. Antibodies

[0040] A. polyclonals

[0041] B. monoclonal

[0042] C. fragments; Kd

[0043] D. anti-idiotypic antibodies

[0044] E. hybridoma cell lines

[0045] VII. Kits, Diagnosis, and Quantitation

[0046] A. ELISA

[0047] B. assay mRNA encoding

[0048] C. qualitative/quantitative

[0049] D. kits

[0050] VIII. Therapeutic compositions, methods

[0051] A. combination compositions

[0052] B. unit dose

[0053] C. administration

[0054] IX. Screening

[0055] I. General

[0056] The present invention provides the amino acid sequence and DNAsequence of mammalian, herein primate, cytokine receptor-like subunitmolecules, this one designated DNAX Cytokine Receptor Subunit 5 (DCRS5)having particular defined properties, both structural and biological.Various cDNAs encoding these molecules were obtained from primate, e.g.,human, cDNA sequence libraries. Other primate or other mammaliancounterparts would also be desired.

[0057] Additionally, the invention provides matching of the p40/IL-B30ligand with receptor subunits DCRS5 and IL-12Rβ1, which pairing providesinsight into indications for use of the agonists and antgonists basedupon reagents directed thereto.

[0058] Some of the standard methods applicable are described orreferenced, e.g., in Maniatis, et al. (1982) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring HarborPress; Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual,(2d ed.), vols. 1 3, CSH Press, NY; Ausubel, et al., Biology, GreenePublishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987 andperiodic supplements) Current Protocols in Molecular Biology,Greene/Wiley, New York; each of which is incorporated herein byreference.

[0059] Nucleotide (SEQ ID NO:1) and corresponding amino acid sequence(SEQ ID NO:2) of a primate, e.g., human, DCRS5 coding segment is shownin Table 1. The predicted signal sequence is indicated, but may dependon cell type, or may be a few residues in either direction. Potential Nglycosylation sites are at Asparagine residues 6, 24, 58, 118, 157, 209,and 250. Disulfide linkages are likely to be found between cysteineresidues at positions 29 and 78; and a conserved C_CXW motif is found atpositions 110/121/123. The tryptophan at 219; and the WxxWS motif from281-285 are notable. The segment from about 1-101 is an Ig domain; fromabout 102-195 is a cytokine binding domain 1; from about 196-297 is acytokine binding domain 2; from about 298-330 is a linker; from about329-354 is a transmembrane segment; and from about 356-606 is anintracellular domain. Intracellular features include putative SH2binding sites at Y374-I377, Y461-Q464, and Y588-Q591; and potentiallyimportant tyrosine residues at 406, 427, 440, and 453. These sites andboundaries are notable.

[0060] The ORF contains a putative signal sequence which is predicted tobe cleaved at . . . CHG/GIT . . . as shown above. A predictedextracellular domain of 328 amino acids is followed by a putativetransmembrane segment, and finally a cytoplasmic domain of about 252amino acids. The ligand-binding functions are predicted to reside in theextracellular domain.

[0061] The reverse translation nucleic acid sequence is provided inTable 2. TABLE 1 Nucleotide and polypeptide sequences of DNAX CytokineReceptor Subunit like embodiments (DCRS5). Primate, e.g., humanembodiment (see SEQ ID NO: 1 and 2). Predicted signal sequenceindicated, but may vary by a few positions and depending upon cell type.Identified positions of variation are at nucleotides 127 and 563; whichare paired G and G (translating to the combination of Q and G) or T andA (translating to H and R). gtggtacggg aattccattg tgttgggcag ccaacaagggtggcagcctg gctctgaagt 60 ggaattatgt gcttcaaaca ggttgaaaga gggaaacagtcttttcctgc ttccagac 118 atg aat cak gtc act att caa tgg gat gca gta atagcc ctt tac ata 166 Met Asn Xaa Val Thr Ile Gln Trp Asp Ala Val Ile AlaLeu Tyr Ile             −20                 −15                 −10 ctcttc agc tgg tgt cat gga gga att aca aat ata aac tgc tct ggc 214 Leu PheSer Trp Cys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly         −5              −1   1               5 cac atc tgg gta gaa ccagcc aca att ttt aag atg ggt atg aat atc 262 His Ile Trp Val Glu Pro AlaThr Ile Phe Lys Met Gly Met Asn Ile 10                  15                  20                  25 tct atatat tgc caa gca gca att aag aac tgc caa cca agg aaa ctt 310 Ser Ile TyrCys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu                 30                  35                  40 cat ttt tataaa aat ggc atc aaa gaa aga ttt caa atc aca agg att 358 His Phe Tyr LysAsn Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile              45                  50                  55 aat aaa aca acagct cgg ctt tgg tat aaa aac ttt ctg gaa cca cat 406 Asn Lys Thr Thr AlaArg Leu Trp Tyr Lys Asn Phe Leu Glu Pro His         60                  65                  70 gct tct atg tac tgcact gct gaa tgt ccc aaa cat ttt caa gag aca 454 Ala Ser Met Tyr Cys ThrAla Glu Cys Pro Lys His Phe Gln Glu Thr     75                  80                  85 ctg ata tgt gga aaa gacatt tct tct gga tat ccg cca gat att cct 502 Leu Ile cys Gly Lys Asp IleSer Ser Gly Tyr Pro Pro Asp Ile Pro 90                  95                 100                 105 gat gaagta acc tgt gtc att tat gaa tat tca ggc aac atg act tgc 550 Asp Glu ValThr Cys Val Ile Tyr Glu Tyr Ser Gly Asn Met Thr Cys                110                 115                 120 acc tgg aatgct rgg aag ctc acc tac ata gac aca aaa tac gtg gta 598 Thr Trp Asn AlaXaa Lys Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val            125                 130                 135 cat gtg aag agttta gag aca gaa gaa gag caa cag tat ctc acc tca 646 His Val Lys Ser LeuGlu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser        140                 145                 150 agc tat att aac atctcc act gat tca tta caa ggt ggc aag aag tac 694 Ser Tyr Ile Asn Ile SerThr Asp Ser Leu Gln Gly Gly Lys Lys Tyr    155                 160                 165 ttg gtt tgg gtc caa gcagca aac gca cta ggc atg gaa gag tca aaa 742 Leu Val Trp Val Gln Ala AlaAsn Ala Leu Gly Met Glu Glu Ser Lys170                 175                 180                 185 caa ctgcaa att cac ctg gat gat ata gtg ata cct tct gca gcc gtc 790 Gln Leu GlnIle His Leu Asp Asp Ile Val Ile Pro Ser Ala Ala Val                190                 195                 200 att tcc agggct gag act ata aat gct aca gtg ccc aag acc ata att 838 Ile Ser Arg AlaGlu Thr Ile Asn Ala Thr Val Pro Lys Thr Ile Ile            205                 210                 215 tat tgg gat agtcaa aca aca att gaa aag gtt tcc tgt gaa atg aga 886 Tyr Trp Asp Ser GlnThr Thr Ile Glu Lys Val Ser Cys Glu Met Arg        220                 225                 230 tac aag gct aca acaaac caa act tgg aat gtt aaa gaa ttt gac acc 934 Tyr Lys Ala Thr Thr AsnGln Thr Trp Asn Val Lys Glu Phe Asp Thr    235                 240                 245 aat ttt aca tat gtg caacag tca gaa ttc tac ttg gag cca aac att 982 Asn Phe Thr Tyr Val Gln GlnSer Glu Phe Tyr Leu Glu Pro Asn Ile250                 255                 260                 265 aag tacgta ttt caa gtg aga tgt caa gaa aca ggc aaa agg tac tgg 1030 Lys Tyr ValPhe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp                270                 275                 280 cag cct tggagt tca ccg ttt ttt cat aaa aca cct gaa aca gtt ccc 1078 Gln Pro Trp SerSer Pro Phe Phe His Lys Thr Pro Glu Thr Val Pro            285                  290                 295 cag gtc aca tcaaaa gca ttc caa cat gac aca tgg aat tct ggg cta 1126 Gln Val Thr Ser LysAla Phe Gln His Asp Thr Trp Asn Ser Gly Leu        300                 305                 310 aca gtt gct tcc atctct aca ggg cac ctt act tct gac aac aga gga 1174 Thr Val Ala Ser Ile SerThr Gly His Leu Thr Ser Asp Asn Arg Gly    315                 320                 325 gac att gga ctt tta ttggga atg atc gtc ttt gct gtt atg ttg tca 1222 Asp Ile Gly Leu Leu Leu GlyMet Ile Val Phe Ala Val Met Leu Ser330                 335                 340                345 att ctttct ttg att ggg ata ttt aac aga tca ttc cga act ggg att 1270 Ile Leu SerLeu Ile Gly Ile Phe Asn Arg Ser Phe Arg Thr Gly Ile                350                 355                 360 aaa aga aggatc tta ttg tta ata cca aag tgg ctt tat gaa gat att 1318 Lys Arg Arg IleLeu Leu Leu Ile Pro Lys Trp Leu Tyr Glu Asp Ile            365                 370                 375 cct aat atg aaaaac agc aat gtt gtg aaa atg cta cag gaa aat agt 1366 Pro Asn Met Lys AsnSer Asn Val Val Lys Met Leu Gln Glu Asn Ser        380                 385                 390 gaa ctt atg aat aataat tcc agt gag cag gtc cta tat gtt gat ccc 1414 Glu Leu Met Asn Asn AsnSer Ser Glu Gln Val Leu Tyr Val Asp Pro    395                 400                 405 atg att aca gag ata aaagaa atc ttc atc cca gaa cac aag cct aca 1462 Met Ile Thr Glu Ile Lys GluIle Phe Ile Pro Glu His Lys Pro Thr410                  415                 420                 425 gac tacaag aag gag aat aca gga ccc ctg gag aca aga gac tac ccg 1510 Asp Tyr LysLys Glu Asn Thr Gly Pro Leu Glu Thr Arg Asp Tyr Pro                430                 435                 440 caa aac tcgcta ttc gac aat act aca gtt gta tat att cct gat ctc 1558 Gln Asn Ser LeuPhe Asp Asn Thr Thr Val Val Tyr Ile Pro Asp Leu            445                 450                 455 aac act gga tataaa ccc caa att tca aat ttt ctg cct gag gga agc 1606 Asn Thr Gly Tyr LysPro Gln Ile Ser Asn Phe Leu Pro Glu Gly Ser        460                 465                 470 cat ctc agc aat aataat gaa att act tcc tta aca ctt aaa cca cca 1654 His Leu Ser Asn Asn AsnGlu Ile Thr Ser Leu Thr Leu Lys Pro Pro    475                 480                 485 gtt gat tcc tta gac tcagga aat aat ccc agg tta caa aag cat cct 1702 Val Asp Ser Leu Asp Ser GlyAsn Asn Pro Arg Leu Gln Lys His Pro490                 495                 500                 505 aat tttgct ttt tct gtt tca agt gtg aat tca cta agc aac aca ata 1750 Asn Phe AlaPhe Ser Val Ser Ser Val Asn Ser Leu Ser Asn Thr Ile                510                 515                 520 ttt ctt ggagaa tta agc ctc ata tta aat caa gga gaa tgc agt tct 1798 Phe Leu Gly GluLeu Ser Leu Ile Leu Asn Gln Gly Glu Cys Ser Ser            525                 530                 535 cct gac ata caaaac tca gta gag gag gaa acc acc atg ctt ttg gaa 1846 Pro Asp Ile Gln AsnSer Val Glu Glu Glu Thr Thr Met Leu Leu Glu        540                 545                 550 aat gat tca ccc agtgaa act att cca gaa cag acc ctg ctt cct gat 1894 Asn Asp Ser Pro Ser GluThr Ile Pro Glu Gln Thr Leu Leu Pro Asp    555                 560                 565 gaa ttt gtc tcc tgt ttgggg atc gtg aat gag gag ttg cca tct att 1942 Glu Phe Val Ser Cys Leu GlyIle Val Asn Glu Glu Leu Pro Ser Ile570                 575                 580                 585 aat acttat ttt cca caa aat att ttg gaa agc cac ttc aat agg att 1990 Asn Thr TyrPhe Pro Gln Asn Ile Leu Glu Ser His Phe Asn Arg Ile                590                 595                 600 tca ctc ttggaa aag tagagctgtg tggtcaaaat caatatgaga aagctgcctt 2045 Ser Leu Leu GluLys             605 gcaatctgaa cttgggtttt ccctgcaata gaaattgaattctgcctctt tttgaaaaaa 2105 atgtattcac atacaaatct tcacatggac acatgttttcatttcccttg gataaatacc 2165 taggtagggg attgctgggc catatgataa gcatatgtttcagttctacc aatcttgttt 2225 ccagagtagt gacatttctg tgctcctacc atcaccatgtaagaattccc gggagctcca 2285 tgccttttta attttagcca ttcttctgcc tmatttcttaaaattagaga attaaggtcc 2345 cgaaggtgga acatgcttca tggtcacaca tacaggcacaaaaacagcat tatgtggacg 2405 cctcatgtat tttttataga gtcaactatt tcctctttattttccctcat tgaaagatgc 2465 aaaacagctc tctattgtgt acagaaaggg taaataatgcaaaatacctg gtagtaaaat 2525 aaatgctgaa aattttcctt taaaatagaa tcattaggccaggcgtggtg gctcatgctt 2585 gtaatcccag cactttggta ggctgaggtr ggtggatcacctgaggtcag gagttcgagt 2645 ccagcctggc caatatgctg aaaccctgtc tctactaaaattacaaaaat tagccggcca 2705 tggtggcagg tgcttgtaat cccagctact tgggaggctgaggcaggaga atcacttgaa 2765 ccaggaaggc agaggttgca ctgagctgag attgtgccactgcactccag cctgggcaac 2825 aagagcaaaa ctctgtctgg aaaaaaaaaa aaaa 2859MN(O/H)VTIQWDAVIALYILFSWCHGGITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNA(G/R)KLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSPFFHKTPETVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGLLLGMIVFAVMLSILSLIGIFNRSFRTGIKRRILLLIPWL YEDIPNMKNSNVVKMLQENSELMNNNSSEQVLYVDPMITEIKEIFIPEHKPTDYKKENTGPLETRDYPQNSLFDNTTVVYIPDLNTGYKPQISNFLPEGSHLSNNNEITSLTLKPPVDSLDSGNNPRLQKHPNFAFSVSSVNSLSNTIFLGELSLILNQGECSSPDIQNSVEEETTMLLENDSPSETIPEQTLLPDEFVSCLGIVNEELPSINTYFPQNILESHFNRISLLEK

[0062] TABLE 2 Reverse Translation of primate, e.g., human, DCRS5 (SEQID NO:3):ATGAAYCAYGTNACNATHCARTGGGAYGCNGTNATHGCNYTNTAYATHYTNTTYWSNTGGTGYCAYGGNGGNATHACNAAYATHAAYTGYWSNGGNCAYATHTGGGTNGARCCNGCNACNATHTTYAARATGGGNATGAAYATHWSNATHTAYTGYCARGCNGCNATHAARAAYTGYCARCCNMGNAARYTNCAYTTYTAYAARAAYGGNATHAARGARMGNTTYCARATHACNMGNATHAAYAARACNACNGCNMGNYTNTGGTAYAARAAYTTYYTNGARCCNCAYGCNWSNATGTAYTGYACNGCNGARTGYCCNAARCAYTTYCARGARACNYTNATHTGYGGNAARGAYATHWSNWSNGGNTAYCCNCCNGAYATHCCNGAYGARGTNACNTGYGTNATHTAYGARTAYWSNGGNAAYATGACNTGYACNTGGAAYGCNMGNAARYTNACNTAYATHGAYACNAARTAYGTNGTNCAYGTNAARWSNYTNGARACNGARGARGARCARCARTAYYTNACNWSNWSNTAYATHAAYATHWSNACNGAYWSNYTNCARGGNGGNAARAARTAYYTNGTNTGGGTNCARGCNGCNAAYGCNYTNGGNATGGARGARWSNAARCARYTNCARATHCAYYTNGAYGAYATHGTNATHCCNWSNGCNGCNGTNATHWSNMGNGCNGARACNATHAAYGCNACNGTNCCNAARACNATHATHTAYTGGGAYWSNCARACNACNATHGARAARGTNWSNTGYGARATGMGNTAYAARGCNACNACNAAYCARACNTGGAAYGTNAARGARTTYGAYACNAAYTTYACNTAYGTNCARCARWSNGARTTYTAYYTNGARCCNAAYATHAARTAYGTNTTYCARGTNMGNTGYCARGARACNGGNAARMGNTAYTGGCARCCNTGGWSNWSNCCNTTYTTYCAYAARACNCCNGARACNGTNCCNCARGTNACNWSNAARGCNTTYCARCAYGAYACNTGGAAYWSNGGNYTNACNGTNGCNWSNATHWSNACNGGNCAYYTNACNWSNGAYAAYMGNGGNGAYATHGGNYTNYTNYTNGGNATGATHGTNTTYGCNGTNATGYTNWSNATHYTNWSNYTNATHGGNATHTTYAAYMGNWSNTTYMGNACNGGNATHAARMGNMGNATHYTNYTNYTNATHCCNAARTGGYTNTAYGARGAYATHCCNAAYATGAARAAYWSNAAYGTNGTNAARATGYTNCARGARAAYWSNGARYTNATGAAYAAYAAYWSNWSNGARCARGTNYTNTAYGTNGAYCCNATGATHACNGARATHAARGARATHTTYATHCCNGARCAYAARCCNACNGAYTAYAARAARGARAAYACNGGNCCNYTNGARACNMGNGAYTAYCCNCARAAYWSNYTNTTYGAYAAYACNACNGTNGTNTAYATHCCNGAYYTNAAYACNGGNTAYAARCCNCARATHWSNAAYTTYYTNCCNGARGGNWSNCAYYTNWSNAAYAAYAAYGARATHACNWSNYTNACNYTNAARCCNCCNGTNGAYWSNYTNGAYWSNGGNAAYAAYCCNMGNYTNCARAARCAYCCNAAYTTYGCNTTYWSNGTNWSNWSNGTNAAYWSNYTNWSNAAYACNATHTTYYTNGGNGARYTNWSNYTNATHYTNAAYCARGGNGARTGYWSNWSNCCNGAYATHCARAAYWSNGTNGARGARGARACNACNATGYTNYTNGARAAYGAYWSNCCNWSNGARACNATHCCNGARCARACNYTNYTNCCNGAYGARTTYGTNWSNTGYYTNGGNATHGTNAAYGARGARYTNCCNWSNATHAAYACNTAYTTYCCNCARAAYATHYTNGARWSNCAYTTYAAYMGNATHWSNYTNYTNGARAAR

[0063] TABLE 3 Alignment of various cytokine receptor subunits. HumanIL-6 receptor pro- tein gp130 is SEQ ID NO: 4 (GenBank M57230); humanIL-12 receptor beta2 subunit is SEQ ID NO: 5 (GenBank U64198). huIL-12R2 1   MAHTFRGCSLAFMFIITWLLIKAKIDACKRGDVTVKPSHVILLGSTVN 48 hugp130 1  MLTLQTWVVQALFIFLTTESTGELLDPCG---YISPESPVVQLHSNFT 45 huDCRS5 1MNHVTIQWDAVIALYILFSWCHGGITNINCS-GHIWVEPATIFKMGMNIS 49            *     .          *         . .  .  . huIL-12R 2 49ITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLG--- 95 hugp130 46AVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIA 95 huDCRS5 50IYCQAAIKN--CQP---RKLHFYKNGIKER-FQITRINKTTARLWYKNFL 93  *    .           .            .   .*   . . huIL-12R 2 96--TTLFVCKLACINSD-EIQTCGAEIPVGVAPEQPQNLSCIQKGEQGTVA 142 hugp130 96SLNIQLTCNILTFGQL-EQNVYGITIISGLPPEKPKNLSCIVN-EGKKMR 143 hnDCRS5 94EPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMT 143       *   .     *  . *  *  *  *. *  ..*.       . huIL-12R 2 143CTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESP 192 hugp130 144CEWDGGRETHLETNFTLKS--EWATHKFADCKAKRDTPTSCTVDYS-TVY 190 huDCRS5 144CTWNARKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGG---- 189* *   . * . * . ..                        . huIL-12R 2 193ESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRC 242 hngp130 191FVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSIL 240 huDCRS5 190-KKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKT 238       * * *.**   *          * * huIL-12R 2 243TLYWRD----EGLVLLNRLRYRPSNSRLNNMVN---VTKAKGRHDLLDLK 285 hugp130 241KLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLK 290 huDCRS5 239IIYWDS--QTTIEKVSCEMRYKATTNQTWNVKEFD-TNFTYVQQSEFYLE 285 . *          .   ..*.      *          .        * huIL-12R 2 286PFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHID 335 hugp130 291PFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSH 340 huDCRS5 286PNIKYVFQVRCQ-ETGKRYWQPWSSPFFHKTPETVP-------------- 320*   * *.. .     *  *  **      * *  * huIL-12R 2 336YS-RQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWT 384 hugp130 341TQGYRTVQLVWKTLPPFEANGKILDYEVT---LTRWKSHLQNYTVNATKL 387 huDCRS5 321-----QVTSKAFQHDTWNSGLTVASISTG------HLTSDN--RGDIGLL 357      .           .   .              .  . huIL-12R 2 385TVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGM 434 hugp130 386TVNLTNDRYLATLTVRNLVGKSDAAVLTIP-ACDFQATRPVMDLKAFPKD 436 huDCRS5 358LGMIVFAVMLSILSLIGIFNRSFRTGIKRR-------------------- 387            ..  .  . *  . . huIL-12R 2 435DNILVTWQPPRKDPSAVQEYVVEWRELHPG-GDTQVPLNWLRSRPYNVSA 483 hugp130 437NMLWVEWTTPRE---SVKKYILEWCVLS---DKAPCITDWQQEDGTVHRT 480 huDCRS5 388----------------ILLLIPKWLYEDIPNMKNSNVVKMLQEN----SE 417                .   .  *                 . huIL-12R 2 484LISENIKSYICYEIRVYALSGDQ-GGCSSILGNSKHKAPLSGPHINAITE 532 hugp130 481YLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKV 530 huDCRS5 418LMNNNSSE--------QVLYVDP-----MITEIKEIFIPEHKPTDYKKE- 453 .  *             .  *       *        *   * huIL-12R 2 533EKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNS 582 hugp130 531GKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGN----ETAVNVDSSHTE 576 huDCRS5 454--NTGPLETRDYP---QNSLFDNTTVVYIPDLNTG------YKPQISN-- 490  .   .     *   *     .  .       .            * * huIL-12R 2 583HPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKAN-WMAFVAPSI 631 hugp130 577YTLSSLTSDTLYMVRMAAYTDEG-GKDGPEFTFTTPKFAQGEIEAIVVPV 625 huDCRS5 491------------------FLPEG--------------------------- 495                      * huIL-12R 2 632CIAIIMVGIFSTHYFQQKVFVLLAALRP-----------QWCSREIPDPA 670 hugp130 626CLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHN 675 huDCRS5 496-----------SHLSNNN-EITSLTLKP--------------PVDSLDSG 519                .   .    . * huIL-12R 2 671NSTCAKKYPIAEEKTQLPLDRLLID-WPTPEDPEPLVIS--EVLHQVTPV 717 hugp130 676FNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHS 725 huDCRS5 520NNPRLQKHPN-FAFSVSSVNSLSNT-------------I---FLGELSLI 552     .            .                        . huIL-12R 2 718FRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPPRALQESRQLVDLY 767 hugp130 726SGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQ 775 huDCRS5 553LNQGECS---S--PDIQNSVEEETTMLLENDSP----------------- 580     .*        *      * huIL-12R 2 768KVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSN---IDDLPSHEAP 814 hugp130 776VFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESS 825 huDCRS5 581--SETIPEQTLLPDEFVSCLGIVNEELPSINTYFPQN---ILESHFNR-- 623    .                     .       * .         . huIL-12R 2 815LADSLEELEPQHISLS-----VFPSSSLHPLTFSCG-------------- 845 hugp130 826PDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAAD 875 huDCRS5 624--ISLLEK 629    *  * huIL-12R 2 846 ----------DKLTLDQLKMRCDSLML 862hugp130 876 AFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 918 huDCRS5 630629

[0064] The closest relatives of the extracellular domain of “IL-30R” arethe IL-6 signal transducer gp130 and IL-12Rβ2. Somewhat less closerelatives are GCSF receptor, leptin receptor, leukemia inhibitory factorreceptor, and CNTF receptor. Thus “IL-30R” is a member of the class Ibranch of the cytokine receptor superfamily and is closely related tothe IL-6R/IL-12R family.

[0065] Table 3 shows comparison of the available sequences of primatereceptor subunits with the primate, e.g., human DCRS5 (IL-30R). TheDCRS5 shows similarity to the IL-6 receptor subunit gp130 (e.g., IL-6Rsubunit) and the IL-12Rβ2 subunit. The DCRS5 exhibits structuralfeatures of a beta subunit, but the actual sequence of proteininteractions and signaling remains unresolved.

[0066] As used herein, the term DCRS5 shall be used to describe aprotein comprising the amino acid sequence shown in Table 1. In manycases, a substantial fragment thereof will be functionally orstructurally equivalent, including, e.g., additional extracellularsegments. The invention also includes a protein variation of therespective DCRS5 allele whose sequence is provided, e.g., a mutein orother construct. Typically, such variants will exhibit less than about10% sequence differences with the target region, and thus will oftenhave between 1- and 11-fold substitutions, e.g., 2-, 3-, 5, 7-fold, andothers. It also encompasses allelic and other variants, e.g., naturalpolymorphisms, of the protein described. Typically, it will bind to itscorresponding biological ligand, perhaps in a dimerized state with analpha receptor subunit, with high affinity, e.g., at least about 100 nM,usually better than about 30 nM, preferably better than about 10 nM, andmore preferably at better than about 3 nM. The term shall also be usedherein to refer to related naturally occurring forms, e.g., alleles,polymorphic variants, and metabolic variants of the mammalian protein.Preferred forms of the receptor complexes will bind the appropriateligand with an affinity and selectivity appropriate for aligand-receptor interaction.

[0067] This invention also encompasses combinations of proteins orpeptides having substantial amino acid sequence identity with the aminoacid sequence in Table 1. It will include sequence variants withrelatively few substitutions, e.g., preferably fewer than about 3-5.

[0068] A substantial polypeptide “fragment”, or “segment”, is a stretchof amino acid residues of at least about 8 amino acids, generally atleast 10 amino acids, more generally at least 12 amino acids, often atleast 14 amino acids, more often at least 16 amino acids, typically atleast 18 amino acids, more typically at least 20 amino acids, usually atleast 22 amino acids, more usually at least 24 amino acids, preferablyat least 26 amino acids, more preferably at least 28 amino acids, and,in particularly preferred embodiments, at least about 30 or more aminoacids. Sequences of segments of different proteins can be compared toone another over appropriate length stretches. In many situations,fragments may exhibit functional properties of the intact subunits,e.g., the extracellular domain of the transmembrane receptor may retainthe ligand binding features, and may be used to prepare a solublereceptor-like complex.

[0069] Amino acid sequence homology, or sequence identity, is determinedby optimizing residue matches. In some comparisons, gaps may beintroduces, as required. See, e.g., Needleham, et al., (1970) J. Mol.Biol. 48:443-453; Sankoff, et al., (1983) chapter one in Time Warps,String Edits, and Macromolecules: The Theory and Practice of SequenceComparison, Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group (GCG), Madison, Wis.; each of which isincorporated herein by reference. This changes when consideringconservative substitutions as matches. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. Homologous amino acid sequences are intended toinclude natural allelic and interspecies variations in the cytokinesequence. Typical homologous proteins or peptides will have from 50 100%homology (if gaps can be introduced), to 60 100% homology (ifconservative substitutions are included) with an amino acid sequencesegment of Table 1. Homology measures will be at least about 70%,generally at least 76%, more generally at least 81%, often at least 85%,more often at least 88%, typically at least 90%, more typically at least92%, usually at least 94%, more usually at least 95%, preferably atleast 96%, and more preferably at least 97%, and in particularlypreferred embodiments, at least 98% or more. The degree of homology willvary with the length of the compared segments. Homologous proteins orpeptides, such as the allelic variants, will share most biologicalactivities with the embodiments described in Table 1, particularly theintracellular portion.

[0070] As used herein, the term “biological activity” is used todescribe, without limitation, effects on signaling, inflammatoryresponses, innate immunity, and/or morphogenic development bycytokine-like ligands. For example, these receptors should mediatephosphatase or phosphorylase activities, which activities are easilymeasured by standard procedures. See, e.g., Hardie, et al. (eds. 1995)The Protein Kinase FactBook vols. I and II, Academic Press, San Diego,Calif.; Hanks, et al. (1991) Meth. Enzymol. 200:38-62; Hunter, et al.(1992) Cell 70:375-388; Lewin (1990) Cell 61:743-752; Pines, et al.(1991) Cold Spring Harbor Symp. Quant. Biol. 56:449-463; and Parker, etal. (1993) Nature 363:736-738. The receptors, or portions thereof, maybe useful as phosphate labeling enzymes to label general or specificsubstrates. The subunits may also be functional immunogens to elicitrecognizing antibodies, or antigens capable of binding antibodies.

[0071] The terms ligand, agonist, antagonist, and analog of, e.g., aDCRS5 petition features of ligand-receptor interactions, e.g., where thereceptor is a natural receptor or an antibody. The cellular responseslikely are typically mediated through receptor tyrosine kinase pathways.

[0072] Also, a ligand is a molecule which serves either as a naturalligand to which said receptor, or an analog thereof, binds, or amolecule which is a functional analog of the natural ligand. Thefunctional analog may be a ligand with structural modifications, or maybe a wholly unrelated molecule which has a molecular shape whichinteracts with the appropriate ligand binding determinants. The ligandsmay serve as agonists or antagonists, see, e.g., Goodman, et al. (eds.1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics,Pergamon Press, New York.

[0073] Rational drug design may also be based upon structural studies ofthe molecular shapes of a receptor or antibody and other effectors orligands. See, e.g., Herz, et al. (1997) J. Recept. Signal Transduct.Res. 17:671-776; and Chaiken, et al. (1996) Trends Biotechnol.14:369-375. Effectors may be other proteins which mediate otherfunctions in response to ligand binding, or other proteins whichnormally interact with the receptor. One means for determining whichsites interact with specific other proteins is a physical structuredetermination, e.g., x-ray crystallography or 2 dimensional NMRtechniques. These will provide guidance as to which amino acid residuesform molecular contact regions. For a detailed description of proteinstructural determination, see, e.g., Blundell and Johnson (1976) ProteinCrystallography, Academic Press, New York, which is hereby incorporatedherein by reference.

[0074] II. Activities

[0075] The cytokine receptor-like proteins will have a number ofdifferent biological activities, e.g., intracellular signaling, e.g.,via STAT4, modulating cell proliferation, or in phosphate metabolism,being added to or removed from specific substrates, typically proteins.Such will generally result in modulation of an inflammatory function,other innate immunity response, or a morphological effect. The subunitwill probably have a specific low affinity binding to the ligand.

[0076] The DCRS5 has the characteristic motifs of a receptor signalingthrough the JAK pathway. See, e.g., Ihle, et al. (1997) Stem Cells15(suppl. 1):105-111; Silvennoinen, et al. (1997) APMIS 105:497-509;Levy (1997) Cytokine Growth Factor Review 8:81-90; Winston and Hunter(1996) Current Biol. 6:668-671; Barrett (1996) Baillieres Clin.Gastroenterol. 10:1-15; and Briscoe, et al. (1996) Philos. Trans. R.Soc. Lond. B. Biol. Sci. 351:167-171. Of particular interest are the SH2binding motifs described above.

[0077] The biological activities of the cytokine receptor subunits willbe related to addition or removal of phosphate moieties to substrates,typically in a specific manner, but occasionally in a non specificmanner. Substrates may be identified, or conditions for enzymaticactivity may be assayed by standard methods, e.g., as described inHardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II,Academic Press, San Diego, Calif.; Hanks, et al. (1991) Meth. Enzymol.200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990) Cell61:743-752;

[0078] Pines, et al. (1991) Cold Spring Harbor Symp. Quant. Biol.56:449-463; and Parker, et al. (1993) Nature 363:736-738.

[0079] The receptor subunits may combine to form functional complexes,e.g., which may be useful for binding ligand or preparing antibodies.These will have substantial diagnostic uses, including detection orquantitation. The functional linkage of the receptor with the p40/IL-B30ligand provides important insights into the clinical indications thatthe receptor will be useful for. Thus, antagonists and agonists willhave predicted functional effects.

[0080] III. Nucleic Acids

[0081] This invention contemplates use of isolated nucleic acid orfragments, e.g., which encode these or closely related proteins, orfragments thereof, e.g., to encode a corresponding polypeptide,preferably one which is biologically active. In addition, this inventioncovers isolated or recombinant DNAs which encode combinations of suchproteins or polypeptides having characteristic sequences, e.g., of theDCRS5s alone or in combination with others such as an IL-12RPβ1 (seeShowe, et al. (1996)) Ann. N.Y. Acad. Sci. 795:413-425; Gately, et al.(1998) Ann. Rev. Immunol. 16:495-521; GenBank U03187, NM_(—)005535)subunit. Typically, the nucleic acid is capable of hybridizing, underappropriate conditions, with a nucleic acid sequence segment shown inTable 1, but preferably not with a corresponding segment of otherreceptors described in Table 3. Said biologically active protein orpolypeptide can be a full length protein, or fragment, and willtypically have a segment of amino acid sequence highly homologous, e.g.,exhibiting significant stretches of identity, to one shown in Table 1.Further, this invention covers the use of isolated or recombinantnucleic acid, or fragments thereof, which encode proteins havingfragments which are equivalent to the DCRS5 proteins, e.g.,intracellular portions. The isolated nucleic acids can have therespective regulatory sequences in the 5′ and 3′ flanks, e.g.,promoters, enhancers, poly-A addition signals, and others from thenatural gene. Combinations, as described, are also provided, e.g.,combining the DCRS5 with the IL-12Rβ1, or their extracellular ligandbinding portions as ligand antagonists. Diagnostic utilities are alsoclearly important, e.g., of polymorphic or other variants.

[0082] An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA,or a mixed polymer, which is substantially pure, e.g., separated fromother components which naturally accompany a native sequence, such asribosomes, polymerases, and flanking genomic sequences from theoriginating species. The term embraces a nucleic acid sequence which hasbeen removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates, which are thereby distinguishablefrom naturally occurring compositions, and chemically synthesizedanalogs or analogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule,either completely or substantially pure.

[0083] An isolated nucleic acid will generally be a homogeneouscomposition of molecules, but will, in some embodiments, containheterogeneity, preferably minor. This heterogeneity is typically foundat the polymer ends or portions not critical to a desired biologicalfunction or activity.

[0084] A “recombinant” nucleic acid is typically defined either by itsmethod of production or its structure. In reference to its method ofproduction, e.g., a product made by a process, the process is use ofrecombinant nucleic acid techniques, e.g., involving human interventionin the nucleotide sequence. Typically this intervention involves invitro manipulation, although under certain circumstances it may involvemore classical animal breeding techniques. Alternatively, it can be anucleic acid made by generating a sequence comprising fusion of twofragments which are not naturally contiguous to each other, but is meantto exclude products of nature, e.g., naturally occurring mutants asfound in their natural state. Thus, e.g., products made by transformingcells with an unnaturally occurring vector is encompassed, as arenucleic acids comprising sequence derived using any syntheticoligonucleotide process. Such a process is often done to replace, e.g.,a codon with a redundant codon encoding the same or a conservative aminoacid, while typically introducing or removing a restriction enzymesequence recognition site, or for some structure-function analysis.Alternatively, the process is performed to join together nucleic acidsegments of desired functions to generate a single genetic entitycomprising a desired combination of functions not found in the commonlyavailable natural forms, e.g., encoding a fusion protein. Restrictionenzyme recognition sites are often the target of such artificialmanipulations, but other site specific targets, e.g., promoters, DNAreplication sites, regulation sequences, control sequences, or otheruseful features may be incorporated by design. A similar concept isintended for a recombinant, e.g., fusion, polypeptide. This will includea dimeric repeat or fusion of the DCRS5 with IL-12Rβ1 subunit.Specifically included are synthetic nucleic acids which, by genetic coderedundancy, encode equivalent polypeptides to fragments of DCRS5 andfusions of sequences from various different related molecules, e.g.,other cytokine receptor family members.

[0085] A “fragment” in a nucleic acid context is a contiguous segment ofat least about 17 nucleotides, generally at least 21 nucleotides, moregenerally at least 25 nucleotides, ordinarily at least 30 nucleotides,more ordinarily at least 35 nucleotides, often at least 39 nucleotides,more often at least 45 nucleotides, typically at least 50 nucleotides,more typically at least 55 nucleotides, usually at least 60 nucleotides,more usually at least 66 nucleotides, preferably at least 72nucleotides, more preferably at least 79 nucleotides, and inparticularly preferred embodiments will be at least 85 or morenucleotides, including 90, 100, 120, 140, 160, 180, 200, etc. Typically,fragments of different genetic sequences can be compared to one anotherover appropriate length stretches, particularly defined segments such asthe domains described below.

[0086] A nucleic acid which codes for the DCRS5 will be particularlyuseful to identify genes, mRNA, and cDNA species which code for itselfor closely related proteins, as well as DNAs which code for polymorphic,allelic, or other genetic variants, e.g., from different individuals orrelated species. Preferred probes for such screens are those regions ofthe receptor which are conserved between different polymorphic variantsor which contain nucleotides which lack specificity, and will preferablybe full length or nearly so. In other situations, polymorphic variantspecific sequences will be more useful. Combinations of polymorphicvariants of DCRS5 with variants of IL- 12Rβ1 may also be diagnosed.

[0087] This invention further covers recombinant nucleic acid moleculesand fragments having a nucleic acid sequence identical to or highlyhomologous to the isolated DNA set forth herein. In particular, thesequences will often be operably linked to DNA segments which controltranscription, translation, and DNA replication. These additionalsegments typically assist in expression of the desired nucleic acidsegment.

[0088] Homologous, or highly identical, nucleic acid sequences, whencompared to one another, e.g., DCRS5 sequences, exhibit significantsimilarity. The standards for homology in nucleic acids are eithermeasures for homology generally used in the art by sequence comparisonor based upon hybridization conditions. Comparative hybridizationconditions are described in greater detail below.

[0089] Substantial identity in the nucleic acid sequence comparisoncontext means either that the segments, or their complementary strands,when compared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 60% of thenucleotides, generally at least 66%, ordinarily at least 71%, often atleast 76%, more often at least 80%, usually at least 84%, more usuallyat least 88%, typically at least 91%, more typically at least about 93%,preferably at least about 95%, more preferably at least about 96 to 98%or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides, including, e.g., segments encoding structural domainsor other segments described. Alternatively, substantial identity willexist when the segments will hybridize under selective hybridizationconditions, to a strand or its complement, typically using a sequencederived from Table 1. Typically, selective hybridization will occur whenthere is at least about 55% homology over a stretch of at least about 14nucleotides, more typically at least about 65%, preferably at leastabout 75%, and more preferably at least about 90%. See, Kanehisa (1984)Nucl. Acids Res. 12:203-213, which is incorporated herein by reference.The length of homology comparison, as described, may be over longerstretches, and in certain embodiments will be over a stretch of at leastabout 17 nucleotides, generally at least about 20 nucleotides,ordinarily at least about 24 nucleotides, usually at least about 28nucleotides, typically at least about 32 nucleotides, more typically atleast about 40 nucleotides, preferably at least about 50 nucleotides,and more preferably at least about 75 to 100 or more nucleotides. Thisincludes, e.g., 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, etc.,and other lengths.

[0090] Stringent conditions, in referring to homology in thehybridization context, will be stringent combined conditions of salt,temperature, organic solvents, and other parameters typically controlledin hybridization reactions. Stringent temperature conditions willusually include temperatures in excess of about 30° C., more usually inexcess of about 37° C., typically in excess of about 45° C., moretypically in excess of about 55° C., preferably in excess of about 65°C., and more preferably in excess of about 70° C. Stringent saltconditions will ordinarily be less than about 500 mM, usually less thanabout 400 mM, more usually less than about 300 mM, typically less thanabout 200 mM, preferably less than about 100 mM, and more preferablyless than about 80 mM, even down to less than about 50 or 20 mM.However, the combination of parameters is much more important than themeasure of any single parameter. See, e.g., Wetmur and Davidson (1968)J. Mol. Biol. 31:349-370, which is hereby incorporated herein byreference.

[0091] The isolated DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, andinversions of nucleotide stretches. These modifications result in novelDNA sequences which encode this protein or its derivatives. Thesemodified sequences can be used to produce mutant proteins (muteins) orto enhance the expression of variant species. Enhanced expression mayinvolve gene amplification, increased transcription, increasedtranslation, and other mechanisms. Such mutant DCRS5 inition of theDCRS5 as set forth above, but having an amino acid sequence whichdiffers from that of other cytokine receptor-like proteins as found innature, whether by way of deletion, substitution, or insertion.

[0092] In particular, “site specific mutant DCRS5” encompasses a proteinhaving substantial sequence identity with a protein of Table 1, andtypically shares most of the biological activities or effects of theforms disclosed herein. Various natural polymorphic variant sequenceswill also be identified.

[0093] Although site specific mutation sites are predetermined, mutantsneed not be site specific. Mammalian DCRS5 mutagenesis can be achievedby making amino acid insertions or deletions in the gene, coupled withexpression. Substitutions, deletions, insertions, or many combinationsmay be generated to arrive at a final construct. Insertions includeamino- or carboxy terminal fusions. Random mutagenesis can be conductedat a target codon and the expressed mammalian DCRS5 mutants can then bescreened for the desired activity, providing some aspect of astructure-activity relationship. Methods for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown in the art, e.g., by M13 primer mutagenesis. See also Sambrook, etal. (1989) and Ausubel, et al. (1987 and periodic Supplements).Particularly useful constructs will be extracellular portions of theDCRS5 associated with IL-12Rβ1 segments.

[0094] The mutations in the DNA normally should not place codingsequences out of reading frames and preferably will not createcomplementary regions that could hybridize to produce secondary mRNAstructure such as loops or hairpins.

[0095] The phosphoramidite method described by Beaucage and Carruthers(1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

[0096] Polymerase chain reaction (PCR) techniques can often be appliedin mutagenesis. Alternatively, mutagenesis primers are commonly usedmethods for generating defined mutations at predetermined sites. See,e.g., Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif.; and Dieffenbach andDveksler (1995; eds.) PCR Primer: A Laboratory Manual Cold Spring HarborPress, CSH, N.Y.

[0097] Certain embodiments of the invention are directed to combinationcompositions comprising the receptor sequences described. In otherembodiments, functional portions of the sequences may be joined toencode fusion proteins. In other forms, variants of the describedsequences may be substituted.

[0098] IV. Proteins, Peptides

[0099] As described above, the present invention encompasses primateDCRS5, e.g., whose sequences are disclosed in Table 1, and describedabove. Allelic and other variants are also contemplated, including,e.g., fusion proteins combining portions of such sequences with others,including, e.g., IL-1 2Rβ1, epitope tags, and functional domains.

[0100] The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these primate or rodentproteins. A heterologous fusion protein is a fusion of proteins orsegments which are naturally not normally fused in the same manner.Thus, the fusion product of a DCRS5 with another cytokine receptor is acontinuous protein molecule having sequences fused in a typical peptidelinkage, typically made as a single translation product and exhibitingproperties, e.g., sequence or antigenicity, derived from each sourcepeptide. A similar concept applies to heterologous nucleic acidsequences. Combinations of various designated proteins into complexesare also provided.

[0101] In addition, new constructs may be made from combining similarfunctional or structural domains from other related proteins, e.g.,cytokine receptors or Toll-like receptors, including species variants.For example, ligand-binding or other segments may be “swapped” betweendifferent new fusion polypeptides or fragments. See, e.g., Cunningham,et al. (1989) Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol.Chem. 263:15985-15992, each of which is incorporated herein byreference. Thus, new chimeric polypeptides exhibiting new combinationsof specificities will result from the functional linkage ofreceptor-binding specificities. For example, the ligand binding domainsfrom other related receptor molecules may be added or substituted forother domains of this or related proteins. The resulting protein willoften have hybrid function and properties. For example, a fusion proteinmay include a targeting domain which may serve to provide sequesteringof the fusion protein to a particular subcellular organelle.

[0102] Candidate fusion partners and sequences can be selected fromvarious sequence data bases, e.g., GenBank, c/o IntelliGenetics,Mountain View, Calif.; and BCG, University of Wisconsin BiotechnologyComputing Group, Madison, Wis., which are each incorporated herein byreference. In particular, combinations of polypeptide sequences providedin Tables 1 and 3 are particularly preferred. Variant forms of theproteins may be substituted in the described combinations.

[0103] The present invention particularly provides muteins which bindcytokine-like ligands, and/or which are affected in signal transduction.Structural alignment of human DCRS5 with other members of the cytokinereceptor family show conserved features/residues. See Table 3. Alignmentof the human DCRS5 sequence with other members of the cytokine receptorfamily indicates various structural and functionally shared features.See also, Bazan, et al. (1996) Nature 379:591; Lodi, et al. (1994)Science 263:1762-1766; Sayle and Milner-White (1995) TIBS 20:374-376;and Gronenberg, et al. (1991) Protein Engineering 4:263-269.

[0104] Substitutions with either mouse sequences or human sequences areparticularly preferred. Conversely, conservative substitutions away fromthe ligand binding interaction regions will probably preserve mostsignaling activities; and conservative substitutions away from theintracellular domains will probably preserve most ligand bindingproperties.

[0105] “Derivatives” of the primate DCRS5 include amino acid sequencemutants, glycosylation variants, metabolic derivatives and covalent oraggregative conjugates with other chemical moieties. Covalentderivatives can be prepared by linkage of functionalities to groupswhich are found in the DCRS5 amino acid side chains or at the N termini,e.g., by means which are well known in the art. These derivatives caninclude, without limitation, aliphatic esters or amides of the carboxylterminus, or of residues containing carboxyl side chains, O acylderivatives of hydroxyl group containing residues, and N acylderivatives of the amino terminal amino acid or amino group containingresidues, e.g., lysine or arginine. Acyl groups are selected from thegroup of alkyl moieties, including C3 to C18 normal alkyl, therebyforming alkanoyl aroyl species.

[0106] In particular, glycosylation alterations are included, e.g., madeby modifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., mammalian glycosylation enzymes. Deglycosylationenzymes are also contemplated. Also embraced are versions of the sameprimary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

[0107] A major group of derivatives are covalent conjugates of thereceptors or fragments thereof with other proteins of polypeptides.These derivatives can be synthesized in recombinant culture such as Nterminal fusions or by the use of agents known in the art for theirusefulness in cross linking proteins through reactive side groups.Preferred derivatization sites with cross linking agents are at freeamino groups, carbohydrate moieties, and cysteine residues.

[0108] Fusion polypeptides between the receptors and other homologous orheterologous proteins are also provided. Homologous polypeptides may befusions between different receptors, resulting in, for instance, ahybrid protein exhibiting binding specificity for multiple differentcytokine ligands, or a receptor which may have broadened or weakenedspecificity of substrate effect. Likewise, heterologous fusions may beconstructed which would exhibit a combination of properties oractivities of the derivative proteins. Typical examples are fusions of areporter polypeptide, e.g., luciferase, with a segment or domain of areceptor, e.g., a ligand-binding segment, so that the presence orlocation of a desired ligand may be easily determined. See, e.g., Dull,et al., U.S. Pat. No. 4,859,609, which is hereby incorporated herein byreference. Other gene fusion partners include glutathione-S-transferase(GST), bacterial β-galactosidase, trpE, Protein A, β-lactamase, alphaamylase, alcohol dehydrogenase, and yeast alpha mating factor. See,e.g., Godowski, et al. (1988) Science 241:812-816. Labeled proteins willoften be substituted in the described combinations of proteins.Associations of the DCRS5 with the IL-12Rβ1 are particularlysignificant, as described.

[0109] The phosphoramidite method described by Beaucage and Carruthers(1981) Tetra, Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

[0110] Such polypeptides may also have amino acid residues which havebeen chemically modified by phosphorylation, sulfonation, biotinylation,or the addition or removal of other moieties, particularly those whichhave molecular shapes similar to phosphate groups. In some embodiments,the modifications will be useful labeling reagents, or serve aspurification targets, e.g., affinity ligands.

[0111] Fusion proteins will typically be made by either recombinantnucleic acid methods or by synthetic polypeptide methods. Techniques fornucleic acid manipulation and expression are described generally, forexample, in Sambrook, et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed.), Vols. 1 3, Cold Spring Harbor Laboratory, and Ausubel,et al. (eds. 1987 and periodic supplements) Current Protocols inMolecular Biology, Greene/Wiley, New York, which are each incorporatedherein by reference. Techniques for synthesis of polypeptides aredescribed, for example, in Merrifield (1963) J. Amer. Chem. Soc.85:2149-2156; Merrifield (1986) Science 232: 341-347; and Atherton, etal. (1989) Solid Phase Peptide Synthesis: A Practical Approach, IRLPress, Oxford; each of which is incorporated herein by reference. Seealso Dawson, et al. (1994) Science 266:776-779 for methods to makelarger polypeptides.

[0112] This invention also contemplates the use of derivatives of aDCRS5 other than variations in amino acid sequence or glycosylation.Such derivatives may involve covalent or aggregative association withchemical moieties. These derivatives generally fall into three classes:(1) salts, (2) side chain and terminal residue covalent modifications,and (3) adsorption complexes, e.g., with cell membranes. Such covalentor aggregative derivatives are useful as immunogens, as reagents inimmunoassays, or in purification methods such as for affinitypurification of a receptor or other binding molecule, e.g., an antibody.For example, a cytokine ligand can be immobilized by covalent bonding toa solid support such as cyanogen bromide activated Sepharose, by methodswhich are well known in the art, or adsorbed onto polyolefin surfaces,with or without glutaraldehyde cross linking, for use in the assay orpurification of a cytokine receptor, antibodies, or other similarmolecules. The ligand can also be labeled with a detectable group, e.g.,radioiodinated by the chloramine T procedure, covalently bound to rareearth chelates, or conjugated to another fluorescent moiety for use indiagnostic assays.

[0113] A combination, e.g., including a DCRS5, of this invention can beused as an immunogen for the production of antisera or antibodiesspecific, e.g., capable of distinguishing between other cytokinereceptor family members, for the combinations described. The complexescan be used to screen monoclonal antibodies or antigen-binding fragmentsprepared by immunization with various forms of impure preparationscontaining the protein. In particular, the term “antibodies” alsoencompasses antigen binding fragments of natural antibodies, e.g., Fab,Fab2, Fv, etc. The purified DCRS5 can also be used as a reagent todetect antibodies generated in response to the presence of elevatedlevels of expression, or immunological disorders which lead to antibodyproduction to the endogenous receptor. Additionally, DCRS5 fragments mayalso serve as immunogens to produce the antibodies of the presentinvention, as described immediately below. For example, this inventioncontemplates antibodies having binding affinity to or being raisedagainst the amino acid sequences shown in Table 1, fragments thereof, orvarious homologous peptides. In particular, this invention contemplatesantibodies having binding affinity to, or having been raised against,specific fragments which are predicted to be, or actually are, exposedat the exterior protein surface of the native DCRS5. Complexes ofcombinations of proteins will also be useful, and antibody preparationsthereto can be made.

[0114] In certain other embodiments, soluble constructs, e.g., of theextracellular ligand binding segments of the DCRS5 with the IL-12Rβ1 maybe binding compositions for the ligand and may be useful as eitherligand antagonists, or as antigens to block ligand mediated signaling.Such may be useful either diagnostically, e.g., for histology labelingfor ligand, or therapeutically, e.g., as ligand antagonists.

[0115] The blocking of physiological response to the receptor ligandsmay result from the inhibition of binding of the ligand to the receptor,likely through competitive inhibition. Thus, in vitro assays of thepresent invention will often use antibodies or antigen binding segmentsof these antibodies, soluble receptor constructs, or fragments attachedto solid phase substrates. These assays will also allow for thediagnostic determination of the effects of either ligand binding regionmutations and modifications, or other mutations and modifications, e.g.,which affect signaling or enzymatic function.

[0116] This invention also contemplates the use of competitive drugscreening assays, e.g., where neutralizing antibodies to the receptorcomplexes or fragments compete with a test compound for binding to aligand or other antibody. In this manner, the neutralizing antibodies orfragments can be used to detect the presence of a polypeptide whichshares one or more binding sites to a receptor and can also be used tooccupy binding sites on a receptor that might otherwise bind a ligand.Soluble receptor constructs combining the extracellular, or ligandbinding, domains of the DCRS5 with the IL-12Rβ1, may be usefulantagonists for competitive binding of p40/IL-B30 ligand.

[0117] V. Making Nucleic Acids and Protein

[0118] DNA which encodes the protein or fragments thereof can beobtained by chemical synthesis, screening cDNA libraries, or byscreening genomic libraries prepared from a wide variety of cell linesor tissue samples. Natural sequences can be isolated using standardmethods and the sequences provided herein, e.g., in Table 1. Otherspecies counterparts can be identified by hybridization techniques, orby various PCR techniques, combined with or by searching in sequencedatabases, e.g., GenBank.

[0119] This DNA can be expressed in a wide variety of host cells for thesynthesis of a full length receptor or fragments which can in turn, forexample, be used to generate polyclonal or monoclonal antibodies; forbinding studies; for construction and expression of modified ligandbinding or kinase/phosphatase domains; and for structure/functionstudies. Variants or fragments can be expressed in host cells that aretransformed or transfected with appropriate expression vectors. Thesemolecules can be substantially free of protein or cellular contaminants,other than those derived from the recombinant host, and therefore areparticularly useful in pharmaceutical compositions when combined with apharmaceutically acceptable carrier and/or diluent. The protein, orportions thereof, may be expressed as fusions with other proteins.Combinations of the described proteins, or nucleic acids encoding them,are particularly interesting.

[0120] Expression vectors are typically self replicating DNA or RNAconstructs containing the desired receptor gene, its fragments, orcombination genes, usually operably linked to suitable genetic controlelements that are recognized in a suitable host cell. These controlelements are capable of effecting expression within a suitable host.Multiple genes may be coordinately expressed, and may be on apolycistronic message. The specific type of control elements necessaryto effect expression will depend upon the eventual host cell used.Generally, the genetic control elements can include a prokaryoticpromoter system or a eukaryotic promoter expression control system, andtypically include a transcriptional promoter, an optional operator tocontrol the onset of transcription, transcription enhancers to elevatethe level of mRNA expression, a sequence that encodes a suitableribosome binding site, and sequences that terminate transcription andtranslation. Expression vectors also usually contain an origin ofreplication that allows the vector to replicate independently of thehost cell.

[0121] The vectors of this invention include those which contain DNAwhich encodes a combination of proteins, as described, or a biologicallyactive equivalent polypeptide. The DNA can be under the control of aviral promoter and can encode a selection marker. This invention furthercontemplates use of such expression vectors which are capable ofexpressing eukaryotic cDNAs coding for such proteins in a prokaryotic oreukaryotic host, where the vector is compatible with the host and wherethe eukaryotic cDNAs are inserted into the vector such that growth ofthe host containing the vector expresses the cDNAs in question. Usually,expression vectors are designed for stable replication in their hostcells or for amplification to greatly increase the total number ofcopies of the desirable gene(s) per cell. It is not always necessary torequire that an expression vector replicate in a host cell, e.g., it ispossible to effect transient expression of the protein or its fragmentsin various hosts using vectors that do not contain a replication originthat is recognized by the host cell. It is also possible to use vectorsthat cause integration of the protein encoding portions into the hostDNA by recombination.

[0122] Vectors, as used herein, comprise plasmids, viruses,bacteriophage, integratable DNA fragments, and other vehicles whichenable the integration of DNA fragments into the genome of the host.Expression vectors are specialized vectors which contain genetic controlelements that effect expression of operably linked genes. Plasmids arethe most commonly used form of vector but all other forms of vectorswhich serve an equivalent function and which are, or become, known inthe art are suitable for use herein. See, e.g., Pouwels, et al. (1985and Supplements) Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.,and Rodriguez, et al. (eds. 1988) Vectors: A Survey of Molecular CloningVectors and Their Uses, Buttersworth, Boston, which are incorporatedherein by reference.

[0123] Transformed cells are cells, preferably mammalian, that have beentransformed or transfected with vectors constructed using recombinantDNA techniques. Transformed host cells usually express the desiredproteins, but for purposes of cloning, amplifying, and manipulating itsDNA, do not need to express the subject proteins. This invention furthercontemplates culturing transformed cells in a nutrient medium, thuspermitting the proteins to accumulate. The proteins can be recovered,either from the culture or, in certain instances, from the culturemedium.

[0124] For purposes of this invention, nucleic sequences are operablylinked when they are functionally related to each other. For example,DNA for a presequence or secretory leader is operably linked to apolypeptide if it is expressed as a preprotein or participates indirecting the polypeptide to the cell membrane or in secretion of thepolypeptide. A promoter is operably linked to a coding sequence if itcontrols the transcription of the polypeptide; a ribosome binding siteis operably linked to a coding sequence if it is positioned to permittranslation. Usually, operably linked means contiguous and in readingframe, however, certain genetic elements such as repressor genes are notcontiguously linked but still bind to operator sequences that in turncontrol expression.

[0125] Suitable host cells include prokaryotes, lower eukaryotes, andhigher eukaryotes. Prokaryotes include both gram negative and grampositive organisms, e.g., E. coli and B. subtilis. Lower eukaryotesinclude yeasts, e.g., S. cerevisiae and Pichia, and species of the genusDictyostelium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

[0126] Prokaryotic host vector systems include a wide variety of vectorsfor many different species. As used herein, E. coli and its vectors willbe used generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express thereceptor or its fragments include, but are not limited to, such vectorsas those containing the lac promoter (pUC series); trp promoter (pBR322trp); Ipp promoter (the pIN series); lambda pP or pR promoters (POTS);or hybrid promoters such as ptac (pDR540). See Brosius, et al. (1988)“Expression Vectors Employing Lambda, and Ipp derived Promoters”, inVectors: A Survey of Molecular Cloning Vectors and Their Uses, (eds.Rodriguez and Denhardt), Buttersworth, Boston, Chapter 10, pp. 205 236,which is incorporated herein by reference.

[0127] Lower eukaryotes, e.g., yeasts and Dictyostelium, may betransformed with DCRS5 sequence containing vectors. For purposes of thisinvention, the most common lower eukaryotic host is the baker's yeast,Saccharomyces cerevisiae. It will be used to generically represent lowereukaryotes although a number of other strains and species are alsoavailable. Yeast vectors typically consist of a replication origin(unless of the integrating type), a selection gene, a promoter, DNAencoding the receptor or its fragments, and sequences for translationtermination, polyadenylation, and transcription termination. Suitableexpression vectors for yeast include such constitutive promoters as 3phosphoglycerate kinase and various other glycolytic enzyme genepromoters or such inducible promoters as the alcohol dehydrogenase 2promoter or metallothionine promoter. Suitable vectors includederivatives of the following types: self replicating low copy number(such as the YRp series), self replicating high copy number (such as theYEp series); integrating types (such as the YIp series), or minichromosomes (such as the YCp series).

[0128] Higher eukaryotic tissue culture cells are normally the preferredhost cells for expression of the functionally active interleukin orreceptor proteins. In principle, many higher eukaryotic tissue culturecell lines are workable, e.g., insect baculovirus expression systems,whether from an invertebrate or vertebrate source. However, mammaliancells are preferred. Transformation or transfection and propagation ofsuch cells has become a routine procedure. Examples of useful cell linesinclude HeLa cells, Chinese hamster ovary (CHO) cell lines, baby ratkidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey(COS) cell lines. Expression vectors for such cell lines usually includean origin of replication, a promoter, a translation initiation site, RNAsplice sites (if genomic DNA is used), a polyadenylation site, and atranscription termination site. These vectors also usually contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as from adenovirus, SV40, parvoviruses, vacciniavirus, or cytomegalovirus. Representative examples of suitableexpression vectors include pCDNA1; pCD, see Okayama, et al. (1985) Mol.Cell Biol. 5:1136 1142; pMClneo PolyA, see Thomas, et al. (1987) Cell51:503 512; and a baculovirus vector such as pAC 373 orpAC 610.

[0129] For secreted proteins and some membrane proteins, an open readingframe usually encodes a polypeptide that consists of a mature orsecreted product covalently linked at its N-terminus to a signalpeptide. The signal peptide is cleaved prior to secretion of the mature,or active, polypeptide. The cleavage site can be predicted with a highdegree of accuracy from empirical rules, e.g., von-Heijne (1986) NucleicAcids Research 14:4683-4690 and Nielsen, et al. (1997) Protein Eng.10:1-12, and the precise amino acid composition of the signal peptideoften does not appear to be critical to its function, e.g., Randall, etal. (1989) Science 243:1156-1159; Kaiser et al. (1987) Science235:312-317. The mature proteins of the invention can be readilydetermined using standard methods.

[0130] It will often be desired to express these polypeptides in asystem which provides a specific or defined glycosylation pattern. Inthis case, the usual pattern will be that provided naturally by theexpression system. However, the pattern will be modifiable by exposingthe polypeptide, e.g., an unglycosylated form, to appropriateglycosylating proteins introduced into a heterologous expression system.For example, the receptor gene may be co-transformed with one or moregenes encoding mammalian or other glycosylating enzymes. Using thisapproach, certain mammalian glycosylation patterns will be achievable inprokaryote or other cells. Expression in prokaryote cells will typicallylead to unglycosylated forms of protein.

[0131] The source of DCRS5 can be a eukaryotic or prokaryotic hostexpressing recombinant DCRS5, such as is described above. The source canalso be a cell line, but other mammalian cell lines are alsocontemplated by this invention, with the preferred cell line being fromthe human species.

[0132] Now that the sequences are known, the primate DCRS5, fragments,or derivatives thereof can be prepared by conventional processes forsynthesizing peptides. These include processes such as are described inStewart and Young (1984) Solid Phase Peptide Synthesis, Pierce ChemicalCo., Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice ofPeptide Synthesis, Springer Verlag, New York; and Bodanszky (1984) ThePrinciples of Peptide Synthesis, Springer Verlag, New York; all of eachwhich are incorporated herein by reference. For example, an azideprocess, an acid chloride process, an acid anhydride process, a mixedanhydride process, an active ester process (for example, p nitrophenylester, N hydroxysuccinimide ester, or cyanomethyl ester), acarbodiimidazole process, an oxidative reductive process, or adicyclohexylcarbodiimide (DCCD) additive process can be used. Solidphase and solution phase syntheses are both applicable to the foregoingprocesses. Similar techniques can be used with partial DCRS5 sequences.

[0133] The DCRS5 proteins, fragments, or derivatives are suitablyprepared in accordance with the above processes as typically employed inpeptide synthesis, generally either by a so called stepwise processwhich comprises condensing an amino acid to the terminal amino acid, oneby one in sequence, or by coupling peptide fragments to the terminalamino acid. Amino groups that are not being used in the couplingreaction typically must be protected to prevent coupling at an incorrectlocation.

[0134] If a solid phase synthesis is adopted, the C terminal amino acidis bound to an insoluble carrier or support through its carboxyl group.The insoluble carrier is not particularly limited as long as it has abinding capability to a reactive carboxyl group. Examples of suchinsoluble carriers include halomethyl resins, such as chloromethyl resinor bromomethyl resin, hydroxymethyl resins, phenol resins, tertalkyloxycarbonylhydrazidated resins, and the like.

[0135] An amino group protected amino acid is bound in sequence throughcondensation of its activated carboxyl group and the reactive aminogroup of the previously formed peptide or chain, to synthesize thepeptide step by step. After synthesizing the complete sequence, thepeptide is split off from the insoluble carrier to produce the peptide.This solid phase approach is generally described by Merrifield, et al.(1963) in J. Am. Chem. Soc. 85:2149 2156, which is incorporated hereinby reference.

[0136] The prepared protein and fragments thereof can be isolated andpurified from the reaction mixture by means of peptide separation, e.g.,by extraction, precipitation, electrophoresis, various forms ofchromatography, immunoaffinity, and the like. The receptors of thisinvention can be obtained in varying degrees of purity depending upondesired uses. Purification can be accomplished by use of the proteinpurification techniques disclosed herein, see below, or by the use ofthe antibodies herein described in methods of immunoabsorbant affinitychromatography. This immunoabsorbant affinity chromatography is carriedout by first linking the antibodies to a solid support and thencontacting the linked antibodies with solubilized lysates of appropriatecells, lysates of other cells expressing the receptor, or lysates orsupernatants of cells producing the protein as a result of DNAtechniques, see below.

[0137] Generally, the purified protein will be at least about 40% pure,ordinarily at least about 50% pure, usually at least about 60% pure,typically at least about 70% pure, more typically at least about 80%pure, preferable at least about 90% pure and more preferably at leastabout 95% pure, and in particular embodiments, 97%-99% or more. Puritywill usually be on a weight basis, but can also be on a molar basis.Different assays will be applied as appropriate. Individual proteins maybe purified and thereafter combined.

[0138] VI. Antibodies

[0139] Antibodies can be raised to the various mammalian, e.g., primateDCRS5 proteins and fragments thereof, both in naturally occurring nativeforms and in their recombinant forms, the difference being thatantibodies to the active receptor are more likely to recognize epitopeswhich are only present in the native conformations. Antibodiesrecognizing epitopes presented by the combination, e.g., functionally,of the DCRS5 with the IL-12Rβ1 are also contemplated. Denatured antigendetection can also be useful in, e.g., Western analysis. Anti-idiotypicantibodies are also contemplated, which would be useful as agonists orantagonists of a natural receptor or an antibody.

[0140] Antibodies, including binding fragments and single chainversions, against predetermined fragments of the protein can be raisedby immunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective protein, or screened for agonistic orantagonistic activity. These monoclonal antibodies will usually bindwith at least a KD of about 1 mM, more usually at least about 300 μM,typically at least about 100 μM, more typically at least about 30 μM,preferably at least about 10 μM, and more preferably at least about 3 μMor better.

[0141] The antibodies, including antigen binding fragments, of thisinvention can have significant diagnostic or therapeutic value. They canbe potent antagonists that bind to the receptor and inhibit binding toligand or inhibit the ability of the receptor to elicit a biologicalresponse, e.g., act on its substrate. They also can be useful as nonneutralizing antibodies and can be coupled to toxins or radionuclides tobind producing cells, or cells localized to the source of theinterleukin. Further, these antibodies can be conjugated to drugs orother therapeutic agents, either directly or indirectly by means of alinker.

[0142] The antibodies of this invention can also be useful in diagnosticapplications. As capture or non neutralizing antibodies, they might bindto the receptor without inhibiting ligand or substrate binding. Asneutralizing antibodies, they can be useful in competitive bindingassays. They will also be useful in detecting or quantifying ligand.They may be used as reagents for Western blot analysis, or forimmunoprecipitation or immunopurification of the respective protein.Likewise, nucleic acids and proteins may be immobilized to solidsubstrates for affinity purification or detection methods. Thesubstrates may be, e.g., solid resin beads or sheets of plastic.

[0143] Protein fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. Mammalian cytokine receptors and fragments may be fused orcovalently linked to a variety of immunogens, such as keyhole limpethemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology,Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962)Specificity of Serological Reactions, Dover Publications, New York; andWilliams, et al. (1967) Methods in Immunology and Immunochemistry, Vol.1, Academic Press, New York; each of which are incorporated herein byreference, for descriptions of methods of preparing polyclonal antisera.A typical method involves hyperimmunization of an animal with anantigen. The blood of the animal is then collected shortly after therepeated immunizations and the gamma globulin is isolated.

[0144] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basicand Clinical Immunology (4th ed.), Lange Medical Publications, LosAltos, Calif., and references cited therein; Harlow and Lane (1988)Antibodies: A Laboratory Manual, CSH Press; Goding (1986) MonoclonalAntibodies: Principles and Practice (2d ed.) Academic Press, New York;and particularly in Kohler and Milstein (1975) in Nature 256: 495 497,which discusses one method of generating monoclonal antibodies. Each ofthese references is incorporated herein by reference. Summarizedbriefly, this method involves injecting an animal with an immunogen. Theanimal is then sacrificed and cells taken from its spleen, which arethen fused with myeloma cells. The result is a hybrid cell or“hybridoma” that is capable of reproducing in vitro. The population ofhybridomas is then screened to isolate individual clones, each of whichsecrete a single antibody species to the immunogen. In this manner, theindividual antibody species obtained are the products of immortalizedand cloned single B cells from the immune animal generated in responseto a specific site recognized on the immunogenic substance.

[0145] Other suitable techniques involve in vitro exposure oflymphocytes to the antigenic polypeptides or alternatively to selectionof libraries of antibodies in phage or similar vectors. See, Huse, etal. (1989) “Generation of a Large Combinatorial Library of theImmunoglobulin Repertoire in Phage Lambda,” Science 246:1275-1281; andWard, et al. (1989) Nature 341:544-546, each of which is herebyincorporated herein by reference. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant or chimericimmunoglobulins may be produced, see Cabilly, U.S. Pat. No. 4,816,567;or made in transgenic mice, see Mendez, et al. (1997) Nature Genetics15:146-156. These references are incorporated herein by reference.

[0146] The antibodies of this invention can also be used for affinitychromatography in isolating the DCRS5 proteins or peptides. Columns canbe prepared where the antibodies are linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysatemay be passed through the column, the column washed, followed byincreasing concentrations of a mild denaturant, whereby the purifiedprotein will be released. Alternatively, the protein may be used topurify antibody. Appropriate cross absorptions or depletions may beapplied.

[0147] The antibodies may also be used to screen expression librariesfor particular expression products. Usually the antibodies used in sucha procedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

[0148] Antibodies raised against a cytokine receptor will also be usedto raise anti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of theprotein or cells which express the protein. They also will be useful asagonists or antagonists of the ligand, which may be competitive receptorinhibitors or substitutes for naturally occurring ligands. Certainantibodies to receptor subunits or combinations may serve as activatingantibodies, which may effect signaling thereby serving, e.g., as ligandagonists.

[0149] A cytokine receptor protein that specifically binds to or that isspecifically immunoreactive with an antibody generated against a definedimmunogen, such as an immunogen consisting of the amino acid sequence ofSEQ ID NO:2, is typically determined in an immunoassay. The immunoassaytypically uses a polyclonal antiserum which was raised, e.g., to aprotein of SEQ ID NO:2. This antiserum is selected to have lowcrossreactivity against other cytokine receptor family members, e.g.,IL-12Rβ2 receptor subunit or IL-6 receptor subunit gp 130, preferablyfrom the same species, and any such crossreactivity is removed byimmunoabsorption prior to use in the immunoassay.

[0150] In order to produce antisera for use in an immunoassay, theprotein, e.g., of SEQ ID NO:2, is isolated as described herein. Forexample, recombinant protein may be produced in a mammalian cell line.An appropriate host, e.g., an inbred strain of mice such as Balb/c, isimmunized with the selected protein, typically using a standardadjuvant, such as Freund's adjuvant, and a standard mouse immunizationprotocol (see Harlow and Lane, supra). Alternatively, a syntheticpeptide derived from the sequences disclosed herein and conjugated to acarrier protein can be used an immunogen. Polyclonal sera are collectedand titered against the immunogen protein in an immunoassay, e.g., asolid phase immunoassay with the immunogen immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁴ or greater are selectedand tested for their cross reactivity against other cytokine receptorfamily members, e.g., gp130 or IL-12Rβ1 using a competitive bindingimmunoassay such as the one described in Harlow and Lane, supra, atpages 570-573. Preferably at least two cytokine receptor family membersare used in this determination. These cytokine receptor family memberscan be produced as recombinant proteins and isolated using standardmolecular biology and protein chemistry techniques as described herein.

[0151] Immunoassays in the competitive binding format can be used forthe crossreactivity determinations. For example, the protein of SEQ IDNO:2 can be immobilized to a solid support. Proteins added to the assaycompete with the binding of the antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theantisera to the immobilized protein is compared to the proteins, e.g.,of gp130 or IL-12Rβ2. The percent crossreactivity for the above proteinsis calculated, using standard calculations. Those antisera with lessthan 10% crossreactivity with each of the proteins listed above areselected and pooled. The cross-reacting antibodies are then removed fromthe pooled antisera by immunoabsorption with the above-listed proteins.

[0152] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein to the immunogen protein (e.g., the DCRS5 like protein of SEQ IDNO:2). In order to make this comparison, the two proteins are eachassayed at a wide range of concentrations and the amount of each proteinrequired to inhibit 50% of the binding of the antisera to theimmobilized protein is determined. If the amount of the second proteinrequired is less than twice the amount of the protein of the selectedprotein or proteins that is required, then the second protein is said tospecifically bind to an antibody generated to the immunogen.

[0153] It is understood that these cytokine receptor proteins aremembers of a family of homologous proteins that comprise many identifiedgenes. For a particular gene product, such as the DCRS5, the term refersnot only to the amino acid sequences disclosed herein, but also to otherproteins that are allelic, non-allelic, or species variants. It is alsounderstood that the terms include nonnatural mutations introduced bydeliberate mutation using conventional recombinant technology such assingle site mutation, or by excising short sections of DNA encoding therespective proteins, or by substituting new amino acids, or adding newamino acids. Such minor alterations typically will substantiallymaintain the immunoidentity of the original molecule and/or itsbiological activity. Thus, these alterations include proteins that arespecifically immunoreactive with a designated naturally occurring DCRS5protein. The biological properties of the altered proteins can bedetermined by expressing the protein in an appropriate cell line andmeasuring the appropriate effect, e.g., upon transfected lymphocytes.

[0154] Particular protein modifications considered minor would includeconservative substitution of amino acids with similar chemicalproperties, as described above for the cytokine receptor family as awhole. By aligning a protein optimally with the protein of the cytokinereceptors and by using the conventional immunoassays described herein todetermine immunoidentity, one can determine the protein compositions ofthe invention.

[0155] Moreover, antibodies against the receptor subunits may serve tosterically block ligand binding to the functional receptor. Suchantibodies may be raised to either subunit alone, or to the combinationof DCRS5 with IL-12Rβ1. Antibody antagonists would result.

[0156] VII. Kits, Diagnosis, and Quantitation

[0157] Both naturally ocurring and recombinant forms of the cytokinereceptor like molecules of this invention are particularly useful inkits and assay methods. For example, these methods would also be appliedto screening for binding activity, e.g., ligands for these proteins.Several methods of automating assays have been developed in recent yearsso as to permit screening of tens of thousands of compounds per year.See, e.g., a BIOMEK automated workstation, Beckman Instruments, PaloAlto, Calif., and Fodor, et al. (1991) Science 251:767-773, which isincorporated herein by reference. The latter describes means for testingbinding by a plurality of defined polymers synthesized on a solidsubstrate. The development of suitable assays to screen for a ligand oragonist/antagonist homologous proteins can be greatly facilitated by theavailability of large amounts of purified, soluble cytokine receptors inan active state such as is provided by this invention.

[0158] Purified DCRS5 can be coated directly onto plates for use in theaforementioned ligand screening techniques. However, non neutralizingantibodies to these proteins can be used as capture antibodies toimmobilize the respective receptor on the solid phase, useful, e.g., indiagnostic uses.

[0159] This invention also contemplates use of DCRS5, fragments thereof,peptides, and their fusion products in a variety of diagnostic kits andmethods for detecting the presence of the protein or its ligand.Alternatively, or additionally, antibodies against the molecules may beincorporated into the kits and methods. Typically the kit will have acompartment containing either a DCRS5 peptide or gene segment or areagent which recognizes one or the other. Typically, recognitionreagents, in the case of peptide, would be a receptor or antibody, or inthe case of a gene segment, would usually be a hybridization probe.Other kit components may include other proteins or reagents related tothe p40, IL-B30, or IL-12Rβ1 polypeptides of the ligand/receptorpairing.

[0160] A preferred kit for determining the concentration of DCRS5 in asample would typically comprise a labeled compound, e.g., ligand orantibody, having known binding affinity for DCRS5, a source of DCRS5(naturally occurring or recombinant) as a positive control, and a meansfor separating the bound from free labeled compound, for example a solidphase for immobilizing the DCRS5 in the test sample. Compartmentscontaining reagents, and instructions, will normally be provided.Appropriate nucleic acid or protein containing kits are also provided.

[0161] Antibodies, including antigen binding fragments, specific formammalian DCRS5 or a peptide fragment, or receptor fragments are usefulin diagnostic applications to detect the presence of elevated levels ofligand and/or its fragments. Diagnostic assays may be homogeneous(without a separation step between free reagent and antibody-antigencomplex) or heterogeneous (with a separation step). Various commercialassays exist, such as radioimmunoassay (RIA), enzyme linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA), enzyme multipliedimmunoassay technique (EMIT), substrate labeled fluorescent immunoassay(SLFIA) and the like. For example, unlabeled antibodies can be employedby using a second antibody which is labeled and which recognizes theantibody to a cytokine receptor or to a particular fragment thereof.These assays have also been extensively discussed in the literature.See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH.,and Coligan (ed. 1991 and periodic supplements) Current Protocols InImmunology Greene/Wiley, New York.

[0162] Anti-idiotypic antibodies may have similar use to serve asagonists or antagonists of cytokine receptors. These should be useful astherapeutic reagents under appropriate circumstances.

[0163] Frequently, the reagents for diagnostic assays are supplied inkits, so as to optimize the sensitivity of the assay. For the subjectinvention, depending upon the nature of the assay, the protocol, and thelabel, either labeled or unlabeled antibody, or labeled ligand isprovided.

[0164] This is usually in conjunction with other additives, such asbuffers, stabilizers, materials necessary for signal production such assubstrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments for each useful reagent, andwill contain instructions for proper use and disposal of reagents.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium havingappropriate concentrations for performing the assay.

[0165] The aforementioned constituents of the diagnostic assays may beused without modification or may be modified in a variety of ways. Forexample, labeling may be achieved by covalently or non covalentlyjoining a moiety which directly or indirectly provides a detectablesignal. In many of these assays, a test compound, cytokine receptor, orantibodies thereto can be labeled either directly or indirectly.Possibilities for direct labeling include label groups: radiolabels suchas ¹²⁵I, enzymes (U.S. Pat. No. 3,645,090) such as peroxidase andalkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475)capable of monitoring the change in fluorescence intensity, wavelengthshift, or fluorescence polarization. Both of the patents areincorporated herein by reference. Possibilities for indirect labelinginclude biotinylation of one constituent followed by binding to avidincoupled to one of the above label groups.

[0166] There are also numerous methods of separating the bound from thefree ligand, or alternatively the bound from the free test compound. Thecytokine receptor can be immobilized on various matrixes followed bywashing. Suitable matrices include plastic such as an ELISA plate,filters, and beads. Methods of immobilizing the receptor to a matrixinclude, without limitation, direct adhesion to plastic, use of acapture antibody, chemical coupling, and biotin avidin. The last step inthis approach involves the precipitation of antibody/antigen complex byany of several methods including those utilizing, e.g., an organicsolvent such as polyethylene glycol or a salt such as ammonium sulfate.Other suitable separation techniques include, without limitation, thefluorescein antibody magnetizable particle method described in Rattle,et al. (1984) Clin. Chem. 30(9):1457 1461, and the double antibodymagnetic particle separation as described in U.S. Pat. No. 4,659,678,each of which is incorporated herein by reference.

[0167] The methods for linking protein or fragments to various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

[0168] Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence ofan cytokine receptor. These sequences can be used as probes fordetecting levels of the respective cytokine receptor in patientssuspected of having an immunological disorder. The preparation of bothRNA and DNA nucleotide sequences, the labeling of the sequences, and thepreferred size of the sequences has received ample description anddiscussion in the literature. Normally an oligonucleotide probe shouldhave at least about 14 nucleotides, usually at least about 18nucleotides, and the polynucleotide probes may be up to severalkilobases. Various labels may be employed, most commonly radionuclides,particularly ³²P. However, other techniques may also be employed, suchas using biotin modified nucleotides for introduction into apolynucleotide. The biotin then serves as the site for binding to avidinor antibodies, which may be labeled with a wide variety of labels, suchas radionuclides, fluorescers, enzymes, or the like. Alternatively,antibodies may be employed which can recognize specific duplexes,including DNA duplexes, RNA duplexes, DNA RNA hybrid duplexes, or DNAprotein duplexes. The antibodies in turn may be labeled and the assaycarried out where the duplex is bound to a surface, so that upon theformation of duplex on the surface, the presence of antibody bound tothe duplex can be detected. The use of probes to the novel anti senseRNA may be carried out in conventional techniques such as nucleic acidhybridization, plus and minus screening, recombinational probing, hybridreleased translation (HRT), and hybrid arrested translation (HART). Thisalso includes amplification techniques such as polymerase chain reaction(PCR).

[0169] Diagnostic kits which also test for the qualitative orquantitative presence of other markers are also contemplated. Diagnosisor prognosis may depend on the combination of multiple indications usedas markers. Thus, kits may test for combinations of markers. See, e.g.,Viallet, et al. (1989) Progress in Growth Factor Res. 1:89-97. Detectionof polymorphic variations, which may reflect functional receptorsignaling differences, may be useful in determining therapeuticstrategy. Variations which reflect greater or lesser response to ligandmay allow subsetting of responsive/non-responsive patient pools.

[0170] VIII. Therapeutic Utility

[0171] This invention provides reagents with significant therapeuticvalue. See, e.g., Levitzki (1996) Curr. Opin. Cell Biol. 8:239-244. Thecytokine receptors (naturally occurring or recombinant), fragmentsthereof, mutein receptors, and antibodies, along with compoundsidentified as having binding affinity to the receptors or antibodies,should be useful in the treatment of conditions exhibiting abnormalexpression of the receptors or their ligands. Such abnormality willtypically be manifested by immunological disorders. See WO 01/18051,which is incorporated herein by reference. Additionally, this inventionshould provide therapeutic value in various diseases or disordersassociated with abnormal expression or abnormal triggering of responseto the ligand. For example, the p40/IL B30 ligand has been suggested tobe involved in development of cell mediated immunity, e.g., anti-tumoractivity, mounting of humoral and cellular immunity, and antiviraleffects. In particular, the ligand appears to activate NK and T cells.Therapy may be combined with IL-18, IL-12, TNF, IFNγ, radiation/chemotherapy, adjuvants, or antitumor, antiviral, or antifungal compounds.

[0172] Conversely, antagonists, which may be combined with antagonistsof TNF, IFNγ, IL-18, or IL-12, or with IL-10 or steroids, may beindicated in chronic Th1 mediated diseases, autoimmunity, or transplantand/or rejection situations, multiple sclerosis, psoriasis, chronicinflammatory conditions, rheumatoid arthritis, osteoarthritis, orinflammatory bowel diseases. Antagonists may take the form of antibodiesagainst the receptor subunits, soluble receptor constructs, or antisensenucleic acids to one or more of the the receptor subunits. The matchingof the p40/IL-B30 ligand with receptor subunits DCRS5 and IL-12Rβ1provides insight into indications for use of the agonists andantagonists.

[0173] Therapeutically, based on the p40/IL-B30 activities described,antagonists of the cytokine may be effected, e.g., by soluble DCRS5,with or without soluble IL-12Rβ1, or antibodies to either receptorsubunit. Antagonists my be useful as inhibitors of undesirable immune orinflammatory responses, to target memory T cells, or in combination withIL-12/IL-12R antagonists, or other anti-inflammatories orimmunosuppressants. Clinical indications may be chronic inflammation ortransplant situations. Various polymorphisms may enhance or decreasereceptor function, and if dominant, might be useful as therapeutics.Identification of such variants may allow subsetting of responsive ornonresponsive patient pools. The reagents may be useful as detecting orlabeling reagents or ablative reagents for memory T cells and/or NKcells.

[0174] Gene therapy may render desired cell populations response top40/IL-B30 ligand, e.g., as adjuvants for tumor immunotherapy, tofacilitate activation of tumor infiltrating lymphocytes, T cells, or NKcells. Antisense strategies may be applied, e.g., to prevent receptorresponsiveness.

[0175] Various abnormal conditions are known in cell types shown toproduce both IL-12 p40 and/or IL-B30 mRNA by Northern blot analysis. SeeBerkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co.,Rahway, N.J.; Thorn, et al. Harrison's Principles of Internal Medicine,McGraw-Hill, N.Y.; and Weatherall, et al. (eds.) Oxford Textbook ofMedicine, Oxford University Press, Oxford. Many other medical conditionsand diseases will be responsive to treatment by an agonist or antagonistprovided herein. See, e.g., Stites and Terr (eds.; 1991) Basic andClinical Immunology Appleton and Lange, Norwalk, Conn.; and Samter, etal. (eds.) Immunological Diseases Little, Brown and Co. Other likelyindications for treatment include bone remodeling, sexual dysfunction,prevention of neurodegenerative diseases, dementia, stress, and others.These problems should be susceptible to prevention or treatment usingcompositions provided herein.

[0176] Recombinant cytokine receptors, muteins, agonist or antagonistantibodies thereto, or antibodies can be purified and then administeredto a patient. These reagents can be combined for therapeutic use withadditional active ingredients, e.g., in conventional pharmaceuticallyacceptable carriers or diluents, along with physiologically innocuousstabilizers and excipients. These combinations can be sterile, e.g.,filtered, and placed into dosage forms as by lyophilization in dosagevials or storage in stabilized aqueous preparations. This invention alsocontemplates use of antibodies or binding fragments thereof which arenot complement binding.

[0177] Ligand screening using cytokine receptor or fragments thereof canbe performed to identify molecules having binding affinity to thereceptors. Subsequent biological assays can then be utilized todetermine if a putative ligand can provide competitive binding, whichcan block intrinsic stimulating activity. Receptor fragments can be usedas a blocker or antagonist in that it blocks the activity of ligand.Likewise, a compound having intrinsic stimulating activity can activatethe receptor and is thus an agonist in that it simulates the activity ofligand, e.g., inducing signaling. This invention further contemplatesthe therapeutic use of antibodies to cytokine receptors as antagonists.

[0178] The quantities of reagents necessary for effective therapy willdepend upon many different factors, including means of administration,target site, reagent physiological life, pharmacological life,physiological state of the patient, and other medicants administered.Thus, treatment dosages should be titrated to optimize safety andefficacy. Typically, dosages used in vitro may provide useful guidancein the amounts useful for in situ administration of these reagents.Animal testing of effective doses for treatment of particular disorderswill provide further predictive indication of human dosage. Variousconsiderations are described, e.g., in Gilman, et al. (eds. 1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8thEd., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.(1990), Mack Publishing Co., Easton, Pa.; each of which is herebyincorporated herein by reference. Methods for administration arediscussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck†& Co., Rahway, N.J. Because of the likely high affinitybinding, or turnover numbers, between a putative ligand and itsreceptors, low dosages of these reagents would be initially expected tobe effective. And the signaling pathway suggests extremely low amountsof ligand may have effect. Thus, dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 pM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or slow release apparatus will often be utilized forcontinuous administration.

[0179] Cytokine receptors, fragments thereof, and antibodies or itsfragments, antagonists, and agonists, may be administered directly tothe host to be treated or, depending on the size of the compounds, itmay be desirable to conjugate them to carrier proteins such as ovalbuminor serum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationscomprise at least one active ingredient, as defined above, together withone or more acceptable carriers thereof. Each carrier must be bothpharmaceutically and physiologically acceptable in the sense of beingcompatible with the other ingredients and not injurious to the patient.Formulations include those suitable for oral, rectal, nasal, orparenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by methods well knownin the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodmanand Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed.,Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.(1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, N.Y.;Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: TabletsDekker, N.Y.; and Lieberman, et al. (eds. 1990) Pharmaceutical DosageForms: Disperse Systems Dekker, N.Y. The therapy of this invention maybe combined with or used in association with other therapeutic agents,particularly agonists or antagonists of other cytokine receptor familymembers.

[0180] IX. Screening

[0181] Drug screening using DCRS5 or fragments thereof can be performedto identify compounds having binding affinity to the receptor subunit,including isolation of associated components. Subsequent biologicalassays can then be utilized to determine if the compound has intrinsicstimulating activity and is therefore a blocker or antagonist in that itblocks the activity of the ligand.

[0182] Moreover, matching of the p40/IL-B30 ligand with the functionalreceptor of DCRS3 with IL-12Rβ1, allows screening for antagonists andagonists with a positive signaling control. Small molecule or antobidyscreening can be done.

[0183] One method of drug screening utilizes eukaryotic or prokaryotichost cells which are stably transformed with recombinant DNA moleculesexpressing the DCRS5 in combination with another cytokine receptorsubunit, e.g., the IL-12Rβ1. The signaling is believed to use STAT4.Cells may be isolated which express a receptor in isolation from otherfunctional receptors. Such cells, either in viable or fixed form, can beused for standard antibody/antigen or ligand/receptor binding assays.See also, Parce, et al. (1989) Science 246:243-247; and Owicki, et al.(1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses. Competitive assays areparticularly useful, where the cells are contacted and incubated with alabeled receptor or antibody having known binding affinity to theligand, such as ¹²⁵I-antibody, and a test sample whose binding affinityto the binding composition is being measured. The bound and free labeledbinding compositions are then separated to assess the degree of ligandbinding. The amount of test compound bound is inversely proportional tothe amount of labeled receptor binding to the known source. Manytechniques can be used to separate bound from free ligand to assess thedegree of ligand binding. This separation step could typically involve aprocedure such as adhesion to filters followed by washing, adhesion toplastic followed by washing, or centrifugation of the cell membranes.Viable cells could also be used to screen for the effects of drugs oncytokine mediated functions, e.g., STAT4 signaling and others. Somedetection methods allow for elimination of a separation step, e.g., aproximity sensitive detection system.

[0184] The broad scope of this invention is best understood withreference to the following examples, which are not intended to limit theinventions to the specific embodiments.

EXAMPLES

[0185] I. General Methods

[0186] Some of the standard methods are described or referenced, e.g.,in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSHPress, NY; or Ausubel, et al. (1987 and Supplements) Current Protocolsin Molecular Biology, Greene/Wiley, New York. Methods for proteinpurification include such methods as ammonium sulfate precipitation,column chromatography, electrophoresis, centrifugation, crystallization,and others. See, e.g., Ausubel, et al. (1987 and periodic supplements);Coligan, et al. (ed. 1996) and periodic supplements, Current ProtocolsIn Protein Science Greene/Wiley, New York; Deutscher (1990) “Guide toProtein Purification” in Methods in Enzymology, vol. 182, and othervolumes in this series; and manufacturer's literature on use of proteinpurification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad,Richmond, Calif. Combination with recombinant techniques allow fusion toappropriate segments, e.g., to a FLAG sequence or an equivalent whichcan be fused via a protease-removable sequence. See, e.g., Hochuli(1990) “Purification of Recombinant Proteins with Metal ChelateAbsorbent” in Setlow (ed.) Genetic Engineering, Principle and Methods12:87-98, Plenum Press, N.Y.; and Crowe, et al. (1992) QIAexpress: TheHigh Level Expression & Protein Purification System QUIAGEN, Inc.,Chatsworth, Calif.

[0187] Computer sequence analysis is performed, e.g., using availablesoftware programs, including those from the GCG (U. Wisconsin) andGenBank sources. Public sequence databases were also used, e.g., fromGenBank and others.

[0188] Many techniques applicable to IL-10 receptors may be applied tothe DCRS5, as described, e.g., in U.S. Pat. No. 5,789,192 (IL-10receptor), which is incorporated herein by reference.

[0189] II. Functional Cloning

[0190] It was observed that anti-hIL-12Rβ1 antibody blocked responses ofhuman T cells to p40/IL-B30, and the p40/IL-B30 bound to IL-12Rβ1. Thissuggested that IL-12Rβ1 was one subunit of the receptor complex forp40/IL-B30.

[0191] A mouse T cell population was identified which responded top40/IL-B30 but not to IL-12, and another population which responded toIL-12 but not p40/IL-B30. In addition, it was observed that Ba/F3 cellsexpressing recombinant mIL-12Rβ1 and mIL-12Rβ2 responded to IL-12, butnot to p40/IL-B30. These results collectively indicated that thereceptor complex for p40/IL-B30 contained the IL-1 2Rβ1 and at least oneother subunit which was not IL-12Rβ2. Accordingly an expression cloningstrategy was devised to isolate this second receptor component.

[0192] A cDNA library was prepared from mRNA isolated from Kit225 cells,an IL-2-dependent human T cell line which responds to both IL-12 andp40/IL-B30. The cDNA library was made using a retroviral expressionvector, pMX. Ba/F3 cells expressing recombinant hIL-12Rβ1 were infectedwith this cDNA library, allowed to recover for 3-4 days in IL-3, thenwashed and plated at ˜15,000 cells/well in 96 well plates in mediumcontaining 50 ng/ml hyper-hp40/hIL-B30. See WO 01/18051. Cultures weresupplemented every ˜5 days with additional hyper-hp40/hIL-B30. Afterapproximately two weeks 5-10% of the wells exhibited cell growth. Cellswere recovered from each well, expanded individually in larger culturesin hyper-hp40/hIL-B30, and tested for growth dependence onhyper-hp40/hIL-B30.

[0193] Cells which were p40/IL-B30-dependent for growth were analyzed byPCR for retroviral cDNA inserts. Out of more than 40 isolates analyzed,all but one contained cDNAs encoding the novel receptor DCRS5. Thiscandidate human cDNA was cloned in an expression vector and transfectedinto Ba/F3 cells expressing hIL-12Rβ1. These cells became responsive top40/IL-B30; thus we concluded that the novel cDNA encoded the desiredDCRS5, functionally an IL-B30 receptor subunit.

[0194] III. Features of Full-length DCRS5; Chromosomal Localization

[0195] The cytoplasmic domain of DCRS5 is not overall closely related toother cytokine receptor cytoplasmic domains, a common observation inthis family of molecules. The cytoplasmic domain contains seven tyrresidues, at least three of which are part of recognizable SH2-bindingmotifs: YEDI, YKPQ, and YFPQ. The YEDI motif is similar to identifiedbinding sites for the tyrosine phosphatase shp2. The latter two motifsare very similar to sequences known to bind Stat1/Stat3, or Stat3,respectively. The YKPQ motif, together with nearby flanking sequences,also resembles to a degree the motifs in Stat4 and IL-12Rβ2 which areknown to bind Stat1-3. This is consistent with preliminary datasuggesting that p40/IL-B30, like IL-12, activates Stat4.

[0196] PCR primers derived from the DCRS5 sequence are used to probe ahuman cDNA library. Sequences may be derived, e.g., from Table 1,preferably those adjacent the ends of sequences. Full length cDNAs forprimate, rodent, or other species DCRS5 are cloned, e.g., by DNAhybridization screening of λgt10 phage. PCR reactions are conductedusing T. aquaticus Taqplus DNA polymerase (Stratagene) under appropriateconditions.

[0197] Chromosome spreads are prepared. In situ hybridization isperformed on chromosome preparations obtained fromphytohemagglutinin-stimulated human lymphocytes cultured for 72 h.5-bromodeoxyuridine was added for the final seven hours of culture (60μg/ml of medium), to ensure a posthybridization chromosomal banding ofgood quality.

[0198] A PCR fragment, amplified with the help of primers, is clonedinto an appropriate vector. The vector is labeled by nick-translationwith ³H. The radiolabeled probe is hybridized to metaphase spreads atfinal concentration of 200 ng/ml of hybridization solution as describedin Mattei, et al. (1985) Hum. Genet. 69:327-331.

[0199] After coating with nuclear track emulsion (KODAK NTB2), slidesare exposed. To avoid any slipping of silver grains during the bandingprocedure, chromosome spreads are first stained with buffered Giemsasolution and metaphase photographed. R-banding is then performed by thefluorochrome-photolysis-Giemsa (FPG) method and metaphasesrephotographed before analysis.

[0200] Similar appropriate methods are used for other species.

[0201] IV. Localization of DCRS5 mRNA

[0202] Human multiple tissue (Cat# 1, 2) and cancer cell line blots(Cat# 7757-1), containing approximately 2 μg of poly (A) ⁺ RNA per lane,are purchased from Clontech (Palo Alto, Calif.). Probes are radiolabeledwith [α-³²P] dATP, e.g., using the Amersham Rediprime random primerlabelling kit (RPN1633). Prehybridization and hybridizations areperformed, e.g., at 65° C. in 0.5 M Na₂HPO₄, 7% SDS, 0.5 M EDTA (pH8.0). High stringency washes are conducted, e.g., at 65° C. with twoinitial washes in 2×SSC, 0.1% SDS for 40 min followed by a subsequentwash in 0.1×SSC, 0.1% SDS for 20 min. Membranes are then exposed at −70°C. to X-Ray film (Kodak) in the presence of intensifying screens. Moredetailed studies by cDNA library Southerns are performed with selectedappropriate human DCRS5 clones to examine their expression inhemopoietic or other cell subsets.

[0203] Alternatively, two appropriate primers are selected from Table 1.RT-PCR is used on an appropriate mRNA sample selected for the presenceof message to produce a cDNA, e.g., a sample which expresses the gene.

[0204] Full length clones may be isolated by hybridization of cDNAlibraries from appropriate tissues pre-selected by PCR signal. Northernblots can be performed.

[0205] Message for genes encoding DCRS5 will be assayed by appropriatetechnology, e.g., PCR, immunoassay, hybridization, or otherwise. Tissueand organ cDNA preparations are available, e.g., from Clontech, MountainView, Calif. Identification of sources of natural expression are useful,as described. And the identification of the functional receptor subunitpairing allows for prediction of what cells express the combination ofreceptor subunits which will result in a physiological responsiveness toeach of the cytokine ligands.

[0206] For mouse distribution, e.g., Southern Analysis can be performed:DNA (5 μg) from a primary amplified cDNA library was digested withappropriate restriction enzymes to release the inserts, run on a 1%agarose gel and transferred to a nylon membrane (Schleicher and Schuell,Keene, N.Y.).

[0207] Samples for mouse mRNA isolation may include: resting mousefibroblastic L cell line (C200); Braf:ER (Braf fusion to estrogenreceptor) transfected cells, control (C201); T cells, TH1 polarized(Mell4 bright, CD4+ cells from spleen, polarized for 7 days with IFN-γand anti IL-4; T200); T cells, TH2 polarized (Mell4 bright, CD4+ cellsfrom spleen, polarized for 7 days with IL-4 and anti-IFN-γ; T201); Tcells, highly TH1 polarized (see Openshaw, et al. (1995) J. Exp. Med.182:1357-1367; activated with anti-CD3 for 2, 6, 16 h pooled; T202); Tcells, highly TH2 polarized (see Openshaw, et al. (1995) J. Exp. Med.182:1357-1367; activated with anti-CD3 for 2, 6, 16 h pooled; T203);CD44-CD25+ pre T cells, sorted from thymus (T204); TH1 T cell cloneD1.1, resting for 3 weeks after last stimulation with antigen (T205);TH1 T cell clone D1.1, 10 μg/ml ConA stimulated 15 h (T206); TH2 T cellclone CDC35, resting for 3 weeks after last stimulation with antigen(T207); TH2 T cell clone CDC35, 10 μg/ml ConA stimulated 15 h (T208);Mell4+ naïve T cells from spleen, resting (T209); Mell4+ T cells,polarized to Th1 with IFN-γ/IL-12/anti-IL-4 for 6, 12, 24 h pooled(T210); Mell4+ T cells, polarized to Th2 with IL-4/anti-IFN-γ for 6, 13,24 h pooled (T211); unstimulated mature B cell leukemia cell line A20(B200); unstimulated B cell line CH12 (B201); unstimulated large B cellsfrom spleen (B202); B cells from total spleen, LPS activated (B203);metrizamide enriched dendritic cells from spleen, resting (D200);dendritic cells from bone marrow, resting (D201); monocyte cell line RAW264.7 activated with LPS 4 h (M200); bone-marrow macrophages derivedwith GM and M-CSF (M201); macrophage cell line J774, resting (M202);macrophage cell line J774+LPS+anti-IL-10 at 0.5, 1, 3, 6, 12 h pooled(M203); macrophage cell line J774+LPS+IL-10 at 0.5, 1, 3, 5, 12 hpooled(M204); aerosol challenged mouse lung tissue, Th2 primers, aerosolOVA challenge 7, 14, 23 h pooled (see Garlisi, et al. (1995) ClinicalImmunology and Immunopathology 75:75-83; X206); Nippostrongulus-infectedlung tissue (see Coffman, et al. (1989) Science 245:308-310; X200);total adult lung, normal (O200); total lung, rag-1 (see Schwarz, et al.(1993) Immunodeficiency 4:249-252; O205); IL-10 K.O. spleen (see Kuhn,et al. (1991) Cell 75:263-274; X201); total adult spleen, normal (O201);total spleen, rag-i (O207); IL-10 K.O. Peyer's patches (O202); totalPeyer's patches, normal (O210); IL-10 K.O. mesenteric lymph nodes(X203); total mesenteric lymph nodes, normal (O211); IL-10 K.O colon(X203); total colon, normal (O212); NOD mouse pancreas (see Makino, etal. (1980) Jikken Dobutsu 29:1-13; X205); total thymus, rag-1 (O208);total kidney, rag-1 (0209); total heart, rag-1 (O202); total brain,rag-1 O203); total testes, rag-1 O204); total liver, rag-1 O206); ratnormal joint tissue O300); and rat arthritic joint tissue (X300).

[0208] Samples for human mRNA isolation may include: peripheral bloodmononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells),resting (T100); peripheral blood mononuclear cells, activated withanti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72, resting(T102); T cell, THO clone Mot 72, activated with anti-CD28 and anti-CD3for 3, 6, 12 h pooled (T103); T cell, THO clone Mot 72, anergic treatedwith specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 cloneHY06, resting (T107); T cell, TH1 clone HY06, activated with anti-CD28and anti-CD3 for 3, 6, 12 h pooled (T1 08); T cell, TH1 clone HY06,anergic treated with specific peptide for 2, 6, 12 h pooled (T1 09); Tcell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935,activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); Tcells CD4+CD45RO− T cells polarized 27 days in anti-CD28, IL-4, and antiIFN-γ, TH2 polarized, activated with anti-CD3 and anti-CD28 4 h (T116);T cell tumor lines Jurkat and Hut78, resting (T117); T cell clones,pooled AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118);T cell random γδ T cell clones, resting (T119); Splenocytes, resting(B100); Splenocytes, activated with anti-CD40 and IL-4 (B101); B cellEBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting (B102);B cell line JY, activated with PMA and ionomycin for 1, 6 h pooled(B103); NK 20 clones pooled, resting (K100); NK 20 clones pooled,activated with PMA and ionomycin for 6 h (K101); NKL clone, derived fromperipheral blood of LGL leukemia patient, IL-2 treated (K106); NKcytotoxic clone 640-A30-1, resting (K107); hematopoietic precursor lineTF 1, activated with PMA and ionomycin for 1, 6 h pooled (C100); U937premonocytic line, resting (M100); U937 premonocytic line, activatedwith PMA and ionomycin for 1, 6 h pooled (M101); elutriated monocytes,activated with LPS, IFNγ, anti-IL-10 for 1, 2, 6, 12, 24 h pooled(M102); elutriated monocytes, activated with LPS, IFNγ, IL-10 for 1, 2,6, 12, 24 h pooled (M103); elutriated monocytes, activated with LPS,IFNγ, anti-IL-10 for 4, 16 h pooled (M106); elutriated monocytes,activated with LPS, IFNγ, IL-10 for 4, 16 h pooled (M107); elutriatedmonocytes, activated LPS for 1 h (M108); elutriated monocytes, activatedLPS for 6 h (M109); DC 70% CD1a+, from CD34+ GM-CSF, TNFα 12 days,resting (D101); DC 70% CD1 a+, from CD34+ GM-CSF, TNFα 12 days,activated with PMA and ionomycin for 1 hr (D102); DC 70% CD1a+, fromCD34+ GM-CSF, TNFα 12 days, activated with PMA and ionomycin for 6 hr(D103); DC 95% CD1a+, from CD34+ GM-CSF, TNFα 12 days FACS sorted,activated with PMA and ionomycin for 1, 6 hr pooled (D104); DC 95%CD14+, ex CD34+ GM-CSF, TNFα 12 days FACS sorted, activated with PMA andionomycin 1, 6 hr pooled (D1 05); DC CD1a+ CD86+, from CD34+ GM-CSF,TNFα 12 days FAC sorted, activated with PMA and ionomycin for 1, 6 hpooled (K106); DC from monocytes GM-CSF, IL-4 5 days, resting (D107); DCfrom monocytes GM-CSF, IL-4 5 days, resting (D1 08); DC from monocytesGM-CSF, IL-4 5 days, activated LPS 4, 16 h pooled (DI 09); DC frommonocytes GM-CSF, IL-4 5 days, activated TNFα, monocyte supe for 4, 16 hpooled (D110); leiomyoma L11 benign tumor (X101); normal myometrium M5(O115); malignant leiomyosarcoma GS 1 (X103); lung fibroblast sarcomaline MRC5, activated with PMA and ionomycin for 1, 6 h pooled (C101);

[0209] kidney epithelial carcinoma cell line CHA, activated with PMA andionomycin for 1, 6 h pooled (C102); kidney fetal 28 wk male (O100); lungfetal 28 wk male (O101); liver fetal 28 wk male (O102); heart fetal 28wk male (O103); brain fetal 28 wk male (O104); gallbladder fetal 28 wkmale O106); small intestine fetal 28 wk male O107); adipose tissue fetal28 wk male (O108); ovary fetal 25 wk female (O109); uterus fetal 25 wkfemale (O 110); testes fetal 28 wk male (O111); spleen fetal 28 wk male(O112); adult placenta 28 wk (O113); and tonsil inflamed, from 12 yearold (X100).

[0210] Similar samples may isolated in other species for evaluation.

[0211] V. Cloning of Species Counterparts of DCRS5

[0212] Various strategies are used to obtain species counterparts of theDCRS5, preferably from other primates or rodents. One method is by crosshybridization using closely related species DNA probes. It may be usefulto go into evolutionarily similar species as intermediate steps. Anothermethod is by using specific PCR primers based on the identification ofblocks of similarity or difference between genes, e.g., areas of highlyconserved or nonconserved polypeptide or nucleotide sequence.

[0213] Database searches may identify similar sequences and allowproduction of appropriate probes.

[0214] VI. Production of Mammalian DCRS5 Protein

[0215] An appropriate, e.g., GST, fusion construct is engineered forexpression, e.g., in E. coli. For example, a mouse IGIF pGex plasmid isconstructed and transformed into E. coli. Freshly transformed cells aregrown, e.g., in LB medium containing 50 μg/ml ampicillin and inducedwith IPTG (Sigma, St. Louis, Mo.). After overnight induction, thebacteria are harvested and the pellets containing the DCRS5 protein areisolated. The pellets are homogenized, e.g., in TE buffer (50 mMTris-base pH 8.0, 10 mM EDTA and 2 mM pefabloc) in 2 liters. Thismaterial is passed through a microfluidizer (Microfluidics, Newton,Mass.) three times. The fluidized supernatant is spun down on a SorvallGS-3 rotor for 1 h at 13,000 rpm. The resulting supernatant containingthe cytokine receptor protein is filtered and passed over aglutathione-SEPHAROSE column equilibrated in 50 mM Tris-base pH 8.0. Thefractions containing the DCRS5-GST fusion protein are pooled andcleaved, e.g., with thrombin (Enzyme Research Laboratories, Inc., SouthBend, IN). The cleaved pool is then passed over a Q-SEPHAROSE columnequilibrated in 50 mM Tris-base. Fractions containing DCRS5 are pooledand diluted in cold distilled H₂O, to lower the conductivity, and passedback over a fresh Q-Sepharose column, alone or in succession with animmunoaffinity antibody column.

[0216] Fractions containing the DCRS5 protein are pooled, aliquoted, andstored in the −70° C. freezer.

[0217] Comparison of the CD spectrum with cytokine receptor protein maysuggest that the protein is correctly folded. See Hazuda, et al. (1969)J. Biol. Chem. 264:1689-1693.

[0218] VII. Preparation of Antibodies Specific for DCRS5

[0219] Inbred Balb/c mice are immunized intraperitoneally withrecombinant forms of the protein, e.g., purified DCRS5 or stabletransfected NIH-3T3 cells. Animals are boosted at appropriate timepoints with protein, with or without additional adjuvant, to furtherstimulate antibody production. Serum is collected, or hybridomasproduced with harvested spleens.

[0220] Alternatively, Balb/c mice are immunized with cells transformedwith the gene or fragments thereof, either endogenous or exogenouscells, or with isolated membranes enriched for expression of theantigen. Serum is collected at the appropriate time, typically afternumerous further administrations. Various gene therapy techniques may beuseful, e.g., in producing protein in situ, for generating an immuneresponse. Serum or antibody preparations may be cross-absorbed orimmunoselected to prepare substantially purified antibodies of definedspecificity and high affinity.

[0221] Monoclonal antibodies may be made. For example, splenocytes arefused with an appropriate fusion partner and hybridomas are selected ingrowth medium by standard procedures. Hybridoma supernatants arescreened for the presence of antibodies which bind to the DCRS5, e.g.,by ELISA or other assay. Antibodies which specifically recognizespecific DCRS5 embodiments may also be selected or prepared.

[0222] In another method, synthetic peptides or purified protein arepresented to an immune system to generate monoclonal or polyclonalantibodies. See, e.g., Coligan (ed. 1991) Current Protocols inImmunology Wiley/Greene; and Harlow and Lane (1989) Antibodies: ALaboratory Manual Cold Spring Harbor Press. In appropriate situations,the binding reagent is either labeled as described above, e.g.,fluorescence or otherwise, or immobilized to a substrate for panningmethods. Nucleic acids may also be introduced into cells in an animal toproduce the antigen, which serves to elicit an immune response. See,e.g., Wang, et al. (1993) Proc. Nat'l. Acad. Sci. 90:4156-4160; Barry,et al. (1994) BioTechniques 16:616-619; and Xiang, et al. (1995)Immunity 2: 129-135.

[0223] VIII. Production of fusion proteins with DCRS5

[0224] Various fusion constructs are made with DCRS5, includingembodiments combining such with IL-12Rβ1 sequence. A portion of theappropriate gene is fused to an epitope tag, e.g., a FLAG tag, or to atwo hybrid system construct. See, e.g., Fields and Song (1989) Nature340:245-246.

[0225] The epitope tag may be used in an expression cloning procedurewith detection with anti-FLAG antibodies to detect a binding partner,e.g., ligand for the respective cytokine receptor. The two hybrid systemmay also be used to isolate proteins which specifically bind to DCRS5.

[0226] IX. Structure Activity Relationship

[0227] Information on the criticality of particular residues isdetermined using standard procedures and analysis. Standard mutagenesisanalysis is performed, e.g., by generating many different variants atdetermined positions, e.g., at the positions identified above, andevaluating biological activities of the variants. This may be performedto the extent of determining positions which modify activity, or tofocus on specific positions to determine the residues which can besubstituted to either retain, block, or modulate biological activity.

[0228] Alternatively, analysis of natural variants can indicate whatpositions tolerate natural mutations. This may result from populationalanalysis of variation among individuals, or across strains or species.Samples from selected individuals are analyzed, e.g., by PCR analysisand sequencing. This allows evaluation of population polymorphisms.

[0229] X. Coexpression of DCRS5 and IL-12Rβ1

[0230] A vector, or vectors, encoding the respective genes may betransfected into a cell. Preferably, such vector will have selectionmarkers to identify which cells have successfully been transformed.Coexpression of the two genes will allow the gene products to properlyassociate to form active receptor complexes.

[0231] Alternatively, use of methods causing association of functionaldimers are available. See, e.g., O'Shea, et al. (1989) Science245:646-648; Kostelny, et al. (1992) J. Immunol. 148:1547-1553; andPatel, et al. (1996) J. Biol. Chem. 271:30386-30391. Expression ofextracellular domains, and physical association, e.g., driven by Fos/Junleucine zipper affinity, will result in ligand binding constructs whichshould act as binding compounds for diagnostic or therapeutic uses.

[0232] All citations herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

[0233] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

1 5 1 2859 DNA Homo sapiens CDS (119)..(2005) 1 gtggtacggg aattccattgtgttgggcag ccaacaaggg tggcagcctg gctctgaagt 60 ggaattatgt gcttcaaacaggttgaaaga gggaaacagt cttttcctgc ttccagac 118 atg aat cak gtc act attcaa tgg gat gca gta ata gcc ctt tac ata 166 Met Asn Xaa Val Thr Ile GlnTrp Asp Ala Val Ile Ala Leu Tyr Ile 1 5 10 15 ctc ttc agc tgg tgt catgga gga att aca aat ata aac tgc tct ggc 214 Leu Phe Ser Trp Cys His GlyGly Ile Thr Asn Ile Asn Cys Ser Gly 20 25 30 cac atc tgg gta gaa cca gccaca att ttt aag atg ggt atg aat atc 262 His Ile Trp Val Glu Pro Ala ThrIle Phe Lys Met Gly Met Asn Ile 35 40 45 tct ata tat tgc caa gca gca attaag aac tgc caa cca agg aaa ctt 310 Ser Ile Tyr Cys Gln Ala Ala Ile LysAsn Cys Gln Pro Arg Lys Leu 50 55 60 cat ttt tat aaa aat ggc atc aaa gaaaga ttt caa atc aca agg att 358 His Phe Tyr Lys Asn Gly Ile Lys Glu ArgPhe Gln Ile Thr Arg Ile 65 70 75 80 aat aaa aca aca gct cgg ctt tgg tataaa aac ttt ctg gaa cca cat 406 Asn Lys Thr Thr Ala Arg Leu Trp Tyr LysAsn Phe Leu Glu Pro His 85 90 95 gct tct atg tac tgc act gct gaa tgt cccaaa cat ttt caa gag aca 454 Ala Ser Met Tyr Cys Thr Ala Glu Cys Pro LysHis Phe Gln Glu Thr 100 105 110 ctg ata tgt gga aaa gac att tct tct ggatat ccg cca gat att cct 502 Leu Ile Cys Gly Lys Asp Ile Ser Ser Gly TyrPro Pro Asp Ile Pro 115 120 125 gat gaa gta acc tgt gtc att tat gaa tattca ggc aac atg act tgc 550 Asp Glu Val Thr Cys Val Ile Tyr Glu Tyr SerGly Asn Met Thr Cys 130 135 140 acc tgg aat gct rgg aag ctc acc tac atagac aca aaa tac gtg gta 598 Thr Trp Asn Ala Xaa Lys Leu Thr Tyr Ile AspThr Lys Tyr Val Val 145 150 155 160 cat gtg aag agt tta gag aca gaa gaagag caa cag tat ctc acc tca 646 His Val Lys Ser Leu Glu Thr Glu Glu GluGln Gln Tyr Leu Thr Ser 165 170 175 agc tat att aac atc tcc act gat tcatta caa ggt ggc aag aag tac 694 Ser Tyr Ile Asn Ile Ser Thr Asp Ser LeuGln Gly Gly Lys Lys Tyr 180 185 190 ttg gtt tgg gtc caa gca gca aac gcacta ggc atg gaa gag tca aaa 742 Leu Val Trp Val Gln Ala Ala Asn Ala LeuGly Met Glu Glu Ser Lys 195 200 205 caa ctg caa att cac ctg gat gat atagtg ata cct tct gca gcc gtc 790 Gln Leu Gln Ile His Leu Asp Asp Ile ValIle Pro Ser Ala Ala Val 210 215 220 att tcc agg gct gag act ata aat gctaca gtg ccc aag acc ata att 838 Ile Ser Arg Ala Glu Thr Ile Asn Ala ThrVal Pro Lys Thr Ile Ile 225 230 235 240 tat tgg gat agt caa aca aca attgaa aag gtt tcc tgt gaa atg aga 886 Tyr Trp Asp Ser Gln Thr Thr Ile GluLys Val Ser Cys Glu Met Arg 245 250 255 tac aag gct aca aca aac caa acttgg aat gtt aaa gaa ttt gac acc 934 Tyr Lys Ala Thr Thr Asn Gln Thr TrpAsn Val Lys Glu Phe Asp Thr 260 265 270 aat ttt aca tat gtg caa cag tcagaa ttc tac ttg gag cca aac att 982 Asn Phe Thr Tyr Val Gln Gln Ser GluPhe Tyr Leu Glu Pro Asn Ile 275 280 285 aag tac gta ttt caa gtg aga tgtcaa gaa aca ggc aaa agg tac tgg 1030 Lys Tyr Val Phe Gln Val Arg Cys GlnGlu Thr Gly Lys Arg Tyr Trp 290 295 300 cag cct tgg agt tca ccg ttt tttcat aaa aca cct gaa aca gtt ccc 1078 Gln Pro Trp Ser Ser Pro Phe Phe HisLys Thr Pro Glu Thr Val Pro 305 310 315 320 cag gtc aca tca aaa gca ttccaa cat gac aca tgg aat tct ggg cta 1126 Gln Val Thr Ser Lys Ala Phe GlnHis Asp Thr Trp Asn Ser Gly Leu 325 330 335 aca gtt gct tcc atc tct acaggg cac ctt act tct gac aac aga gga 1174 Thr Val Ala Ser Ile Ser Thr GlyHis Leu Thr Ser Asp Asn Arg Gly 340 345 350 gac att gga ctt tta ttg ggaatg atc gtc ttt gct gtt atg ttg tca 1222 Asp Ile Gly Leu Leu Leu Gly MetIle Val Phe Ala Val Met Leu Ser 355 360 365 att ctt tct ttg att ggg atattt aac aga tca ttc cga act ggg att 1270 Ile Leu Ser Leu Ile Gly Ile PheAsn Arg Ser Phe Arg Thr Gly Ile 370 375 380 aaa aga agg atc tta ttg ttaata cca aag tgg ctt tat gaa gat att 1318 Lys Arg Arg Ile Leu Leu Leu IlePro Lys Trp Leu Tyr Glu Asp Ile 385 390 395 400 cct aat atg aaa aac agcaat gtt gtg aaa atg cta cag gaa aat agt 1366 Pro Asn Met Lys Asn Ser AsnVal Val Lys Met Leu Gln Glu Asn Ser 405 410 415 gaa ctt atg aat aat aattcc agt gag cag gtc cta tat gtt gat ccc 1414 Glu Leu Met Asn Asn Asn SerSer Glu Gln Val Leu Tyr Val Asp Pro 420 425 430 atg att aca gag ata aaagaa atc ttc atc cca gaa cac aag cct aca 1462 Met Ile Thr Glu Ile Lys GluIle Phe Ile Pro Glu His Lys Pro Thr 435 440 445 gac tac aag aag gag aataca gga ccc ctg gag aca aga gac tac ccg 1510 Asp Tyr Lys Lys Glu Asn ThrGly Pro Leu Glu Thr Arg Asp Tyr Pro 450 455 460 caa aac tcg cta ttc gacaat act aca gtt gta tat att cct gat ctc 1558 Gln Asn Ser Leu Phe Asp AsnThr Thr Val Val Tyr Ile Pro Asp Leu 465 470 475 480 aac act gga tat aaaccc caa att tca aat ttt ctg cct gag gga agc 1606 Asn Thr Gly Tyr Lys ProGln Ile Ser Asn Phe Leu Pro Glu Gly Ser 485 490 495 cat ctc agc aat aataat gaa att act tcc tta aca ctt aaa cca cca 1654 His Leu Ser Asn Asn AsnGlu Ile Thr Ser Leu Thr Leu Lys Pro Pro 500 505 510 gtt gat tcc tta gactca gga aat aat ccc agg tta caa aag cat cct 1702 Val Asp Ser Leu Asp SerGly Asn Asn Pro Arg Leu Gln Lys His Pro 515 520 525 aat ttt gct ttt tctgtt tca agt gtg aat tca cta agc aac aca ata 1750 Asn Phe Ala Phe Ser ValSer Ser Val Asn Ser Leu Ser Asn Thr Ile 530 535 540 ttt ctt gga gaa ttaagc ctc ata tta aat caa gga gaa tgc agt tct 1798 Phe Leu Gly Glu Leu SerLeu Ile Leu Asn Gln Gly Glu Cys Ser Ser 545 550 555 560 cct gac ata caaaac tca gta gag gag gaa acc acc atg ctt ttg gaa 1846 Pro Asp Ile Gln AsnSer Val Glu Glu Glu Thr Thr Met Leu Leu Glu 565 570 575 aat gat tca cccagt gaa act att cca gaa cag acc ctg ctt cct gat 1894 Asn Asp Ser Pro SerGlu Thr Ile Pro Glu Gln Thr Leu Leu Pro Asp 580 585 590 gaa ttt gtc tcctgt ttg ggg atc gtg aat gag gag ttg cca tct att 1942 Glu Phe Val Ser CysLeu Gly Ile Val Asn Glu Glu Leu Pro Ser Ile 595 600 605 aat act tat tttcca caa aat att ttg gaa agc cac ttc aat agg att 1990 Asn Thr Tyr Phe ProGln Asn Ile Leu Glu Ser His Phe Asn Arg Ile 610 615 620 tca ctc ttg gaaaag tagagctgtg tggtcaaaat caatatgaga aagctgcctt 2045 Ser Leu Leu Glu Lys625 gcaatctgaa cttgggtttt ccctgcaata gaaattgaat tctgcctctt tttgaaaaaa2105 atgtattcac atacaaatct tcacatggac acatgttttc atttcccttg gataaatacc2165 taggtagggg attgctgggc catatgataa gcatatgttt cagttctacc aatcttgttt2225 ccagagtagt gacatttctg tgctcctacc atcaccatgt aagaattccc gggagctcca2285 tgccttttta attttagcca ttcttctgcc tmatttctta aaattagaga attaaggtcc2345 cgaaggtgga acatgcttca tggtcacaca tacaggcaca aaaacagcat tatgtggacg2405 cctcatgtat tttttataga gtcaactatt tcctctttat tttccctcat tgaaagatgc2465 aaaacagctc tctattgtgt acagaaaggg taaataatgc aaaatacctg gtagtaaaat2525 aaatgctgaa aattttcctt taaaatagaa tcattaggcc aggcgtggtg gctcatgctt2585 gtaatcccag cactttggta ggctgaggtr ggtggatcac ctgaggtcag gagttcgagt2645 ccagcctggc caatatgctg aaaccctgtc tctactaaaa ttacaaaaat tagccggcca2705 tggtggcagg tgcttgtaat cccagctact tgggaggctg aggcaggaga atcacttgaa2765 ccaggaaggc agaggttgca ctgagctgag attgtgccac tgcactccag cctgggcaac2825 aagagcaaaa ctctgtctgg aaaaaaaaaa aaaa 2859 2 629 PRT Homo sapiensmisc_feature (3)..(3) The ′Xaa′ at location 3 stands for Gln, or His. 2Met Asn Xaa Val Thr Ile Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile 1 5 1015 Leu Phe Ser Trp Cys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly 20 2530 His Ile Trp Val Glu Pro Ala Thr Ile Phe Lys Met Gly Met Asn Ile 35 4045 Ser Ile Tyr Cys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu 50 5560 His Phe Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile 65 7075 80 Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys Asn Phe Leu Glu Pro His 8590 95 Ala Ser Met Tyr Cys Thr Ala Glu Cys Pro Lys His Phe Gln Glu Thr100 105 110 Leu Ile Cys Gly Lys Asp Ile Ser Ser Gly Tyr Pro Pro Asp IlePro 115 120 125 Asp Glu Val Thr Cys Val Ile Tyr Glu Tyr Ser Gly Asn MetThr Cys 130 135 140 Thr Trp Asn Ala Xaa Lys Leu Thr Tyr Ile Asp Thr LysTyr Val Val 145 150 155 160 His Val Lys Ser Leu Glu Thr Glu Glu Glu GlnGln Tyr Leu Thr Ser 165 170 175 Ser Tyr Ile Asn Ile Ser Thr Asp Ser LeuGln Gly Gly Lys Lys Tyr 180 185 190 Leu Val Trp Val Gln Ala Ala Asn AlaLeu Gly Met Glu Glu Ser Lys 195 200 205 Gln Leu Gln Ile His Leu Asp AspIle Val Ile Pro Ser Ala Ala Val 210 215 220 Ile Ser Arg Ala Glu Thr IleAsn Ala Thr Val Pro Lys Thr Ile Ile 225 230 235 240 Tyr Trp Asp Ser GlnThr Thr Ile Glu Lys Val Ser Cys Glu Met Arg 245 250 255 Tyr Lys Ala ThrThr Asn Gln Thr Trp Asn Val Lys Glu Phe Asp Thr 260 265 270 Asn Phe ThrTyr Val Gln Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile 275 280 285 Lys TyrVal Phe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp 290 295 300 GlnPro Trp Ser Ser Pro Phe Phe His Lys Thr Pro Glu Thr Val Pro 305 310 315320 Gln Val Thr Ser Lys Ala Phe Gln His Asp Thr Trp Asn Ser Gly Leu 325330 335 Thr Val Ala Ser Ile Ser Thr Gly His Leu Thr Ser Asp Asn Arg Gly340 345 350 Asp Ile Gly Leu Leu Leu Gly Met Ile Val Phe Ala Val Met LeuSer 355 360 365 Ile Leu Ser Leu Ile Gly Ile Phe Asn Arg Ser Phe Arg ThrGly Ile 370 375 380 Lys Arg Arg Ile Leu Leu Leu Ile Pro Lys Trp Leu TyrGlu Asp Ile 385 390 395 400 Pro Asn Met Lys Asn Ser Asn Val Val Lys MetLeu Gln Glu Asn Ser 405 410 415 Glu Leu Met Asn Asn Asn Ser Ser Glu GlnVal Leu Tyr Val Asp Pro 420 425 430 Met Ile Thr Glu Ile Lys Glu Ile PheIle Pro Glu His Lys Pro Thr 435 440 445 Asp Tyr Lys Lys Glu Asn Thr GlyPro Leu Glu Thr Arg Asp Tyr Pro 450 455 460 Gln Asn Ser Leu Phe Asp AsnThr Thr Val Val Tyr Ile Pro Asp Leu 465 470 475 480 Asn Thr Gly Tyr LysPro Gln Ile Ser Asn Phe Leu Pro Glu Gly Ser 485 490 495 His Leu Ser AsnAsn Asn Glu Ile Thr Ser Leu Thr Leu Lys Pro Pro 500 505 510 Val Asp SerLeu Asp Ser Gly Asn Asn Pro Arg Leu Gln Lys His Pro 515 520 525 Asn PheAla Phe Ser Val Ser Ser Val Asn Ser Leu Ser Asn Thr Ile 530 535 540 PheLeu Gly Glu Leu Ser Leu Ile Leu Asn Gln Gly Glu Cys Ser Ser 545 550 555560 Pro Asp Ile Gln Asn Ser Val Glu Glu Glu Thr Thr Met Leu Leu Glu 565570 575 Asn Asp Ser Pro Ser Glu Thr Ile Pro Glu Gln Thr Leu Leu Pro Asp580 585 590 Glu Phe Val Ser Cys Leu Gly Ile Val Asn Glu Glu Leu Pro SerIle 595 600 605 Asn Thr Tyr Phe Pro Gln Asn Ile Leu Glu Ser His Phe AsnArg Ile 610 615 620 Ser Leu Leu Glu Lys 625 3 1887 PRT Homo sapiens 3Ala Thr Gly Ala Ala Tyr Cys Ala Tyr Gly Thr Asn Ala Cys Asn Ala 1 5 1015 Thr His Cys Ala Arg Thr Gly Gly Gly Ala Tyr Gly Cys Asn Gly Thr 20 2530 Asn Ala Thr His Gly Cys Asn Tyr Thr Asn Thr Ala Tyr Ala Thr His 35 4045 Tyr Thr Asn Thr Thr Tyr Trp Ser Asn Thr Gly Gly Thr Gly Tyr Cys 50 5560 Ala Tyr Gly Gly Asn Gly Gly Asn Ala Thr His Ala Cys Asn Ala Ala 65 7075 80 Tyr Ala Thr His Ala Ala Tyr Thr Gly Tyr Trp Ser Asn Gly Gly Asn 8590 95 Cys Ala Tyr Ala Thr His Thr Gly Gly Gly Thr Asn Gly Ala Arg Cys100 105 110 Cys Asn Gly Cys Asn Ala Cys Asn Ala Thr His Thr Thr Tyr AlaAla 115 120 125 Arg Ala Thr Gly Gly Gly Asn Ala Thr Gly Ala Ala Tyr AlaThr His 130 135 140 Trp Ser Asn Ala Thr His Thr Ala Tyr Thr Gly Tyr CysAla Arg Gly 145 150 155 160 Cys Asn Gly Cys Asn Ala Thr His Ala Ala ArgAla Ala Tyr Thr Gly 165 170 175 Tyr Cys Ala Arg Cys Cys Asn Met Gly AsnAla Ala Arg Tyr Thr Asn 180 185 190 Cys Ala Tyr Thr Thr Tyr Thr Ala TyrAla Ala Arg Ala Ala Tyr Gly 195 200 205 Gly Asn Ala Thr His Ala Ala ArgGly Ala Arg Met Gly Asn Thr Thr 210 215 220 Tyr Cys Ala Arg Ala Thr HisAla Cys Asn Met Gly Asn Ala Thr His 225 230 235 240 Ala Ala Tyr Ala AlaArg Ala Cys Asn Ala Cys Asn Gly Cys Asn Met 245 250 255 Gly Asn Tyr ThrAsn Thr Gly Gly Thr Ala Tyr Ala Ala Arg Ala Ala 260 265 270 Tyr Thr ThrTyr Tyr Thr Asn Gly Ala Arg Cys Cys Asn Cys Ala Tyr 275 280 285 Gly CysAsn Trp Ser Asn Ala Thr Gly Thr Ala Tyr Thr Gly Tyr Ala 290 295 300 CysAsn Gly Cys Asn Gly Ala Arg Thr Gly Tyr Cys Cys Asn Ala Ala 305 310 315320 Arg Cys Ala Tyr Thr Thr Tyr Cys Ala Arg Gly Ala Arg Ala Cys Asn 325330 335 Tyr Thr Asn Ala Thr His Thr Gly Tyr Gly Gly Asn Ala Ala Arg Gly340 345 350 Ala Tyr Ala Thr His Trp Ser Asn Trp Ser Asn Gly Gly Asn ThrAla 355 360 365 Tyr Cys Cys Asn Cys Cys Asn Gly Ala Tyr Ala Thr His CysCys Asn 370 375 380 Gly Ala Tyr Gly Ala Arg Gly Thr Asn Ala Cys Asn ThrGly Tyr Gly 385 390 395 400 Thr Asn Ala Thr His Thr Ala Tyr Gly Ala ArgThr Ala Tyr Trp Ser 405 410 415 Asn Gly Gly Asn Ala Ala Tyr Ala Thr GlyAla Cys Asn Thr Gly Tyr 420 425 430 Ala Cys Asn Thr Gly Gly Ala Ala TyrGly Cys Asn Met Gly Asn Ala 435 440 445 Ala Arg Tyr Thr Asn Ala Cys AsnThr Ala Tyr Ala Thr His Gly Ala 450 455 460 Tyr Ala Cys Asn Ala Ala ArgThr Ala Tyr Gly Thr Asn Gly Thr Asn 465 470 475 480 Cys Ala Tyr Gly ThrAsn Ala Ala Arg Trp Ser Asn Tyr Thr Asn Gly 485 490 495 Ala Arg Ala CysAsn Gly Ala Arg Gly Ala Arg Gly Ala Arg Cys Ala 500 505 510 Arg Cys AlaArg Thr Ala Tyr Tyr Thr Asn Ala Cys Asn Trp Ser Asn 515 520 525 Trp SerAsn Thr Ala Tyr Ala Thr His Ala Ala Tyr Ala Thr His Trp 530 535 540 SerAsn Ala Cys Asn Gly Ala Tyr Trp Ser Asn Tyr Thr Asn Cys Ala 545 550 555560 Arg Gly Gly Asn Gly Gly Asn Ala Ala Arg Ala Ala Arg Thr Ala Tyr 565570 575 Tyr Thr Asn Gly Thr Asn Thr Gly Gly Gly Thr Asn Cys Ala Arg Gly580 585 590 Cys Asn Gly Cys Asn Ala Ala Tyr Gly Cys Asn Tyr Thr Asn GlyGly 595 600 605 Asn Ala Thr Gly Gly Ala Arg Gly Ala Arg Trp Ser Asn AlaAla Arg 610 615 620 Cys Ala Arg Tyr Thr Asn Cys Ala Arg Ala Thr His CysAla Tyr Tyr 625 630 635 640 Thr Asn Gly Ala Tyr Gly Ala Tyr Ala Thr HisGly Thr Asn Ala Thr 645 650 655 His Cys Cys Asn Trp Ser Asn Gly Cys AsnGly Cys Asn Gly Thr Asn 660 665 670 Ala Thr His Trp Ser Asn Met Gly AsnGly Cys Asn Gly Ala Arg Ala 675 680 685 Cys Asn Ala Thr His Ala Ala TyrGly Cys Asn Ala Cys Asn Gly Thr 690 695 700 Asn Cys Cys Asn Ala Ala ArgAla Cys Asn Ala Thr His Ala Thr His 705 710 715 720 Thr Ala Tyr Thr GlyGly Gly Ala Tyr Trp Ser Asn Cys Ala Arg Ala 725 730 735 Cys Asn Ala CysAsn Ala Thr His Gly Ala Arg Ala Ala Arg Gly Thr 740 745 750 Asn Trp SerAsn Thr Gly Tyr Gly Ala Arg Ala Thr Gly Met Gly Asn 755 760 765 Thr AlaTyr Ala Ala Arg Gly Cys Asn Ala Cys Asn Ala Cys Asn Ala 770 775 780 AlaTyr Cys Ala Arg Ala Cys Asn Thr Gly Gly Ala Ala Tyr Gly Thr 785 790 795800 Asn Ala Ala Arg Gly Ala Arg Thr Thr Tyr Gly Ala Tyr Ala Cys Asn 805810 815 Ala Ala Tyr Thr Thr Tyr Ala Cys Asn Thr Ala Tyr Gly Thr Asn Cys820 825 830 Ala Arg Cys Ala Arg Trp Ser Asn Gly Ala Arg Thr Thr Tyr ThrAla 835 840 845 Tyr Tyr Thr Asn Gly Ala Arg Cys Cys Asn Ala Ala Tyr AlaThr His 850 855 860 Ala Ala Arg Thr Ala Tyr Gly Thr Asn Thr Thr Tyr CysAla Arg Gly 865 870 875 880 Thr Asn Met Gly Asn Thr Gly Tyr Cys Ala ArgGly Ala Arg Ala Cys 885 890 895 Asn Gly Gly Asn Ala Ala Arg Met Gly AsnThr Ala Tyr Thr Gly Gly 900 905 910 Cys Ala Arg Cys Cys Asn Thr Gly GlyTrp Ser Asn Trp Ser Asn Cys 915 920 925 Cys Asn Thr Thr Tyr Thr Thr TyrCys Ala Tyr Ala Ala Arg Ala Cys 930 935 940 Asn Cys Cys Asn Gly Ala ArgAla Cys Asn Gly Thr Asn Cys Cys Asn 945 950 955 960 Cys Ala Arg Gly ThrAsn Ala Cys Asn Trp Ser Asn Ala Ala Arg Gly 965 970 975 Cys Asn Thr ThrTyr Cys Ala Arg Cys Ala Tyr Gly Ala Tyr Ala Cys 980 985 990 Asn Thr GlyGly Ala Ala Tyr Trp Ser Asn Gly Gly Asn Tyr Thr Asn 995 1000 1005 AlaCys Asn Gly Thr Asn Gly Cys Asn Trp Ser Asn Ala Thr His 1010 1015 1020Trp Ser Asn Ala Cys Asn Gly Gly Asn Cys Ala Tyr Tyr Thr Asn 1025 10301035 Ala Cys Asn Trp Ser Asn Gly Ala Tyr Ala Ala Tyr Met Gly Asn 10401045 1050 Gly Gly Asn Gly Ala Tyr Ala Thr His Gly Gly Asn Tyr Thr Asn1055 1060 1065 Tyr Thr Asn Tyr Thr Asn Gly Gly Asn Ala Thr Gly Ala ThrHis 1070 1075 1080 Gly Thr Asn Thr Thr Tyr Gly Cys Asn Gly Thr Asn AlaThr Gly 1085 1090 1095 Tyr Thr Asn Trp Ser Asn Ala Thr His Tyr Thr AsnTrp Ser Asn 1100 1105 1110 Tyr Thr Asn Ala Thr His Gly Gly Asn Ala ThrHis Thr Thr Tyr 1115 1120 1125 Ala Ala Tyr Met Gly Asn Trp Ser Asn ThrThr Tyr Met Gly Asn 1130 1135 1140 Ala Cys Asn Gly Gly Asn Ala Thr HisAla Ala Arg Met Gly Asn 1145 1150 1155 Met Gly Asn Ala Thr His Tyr ThrAsn Tyr Thr Asn Tyr Thr Asn 1160 1165 1170 Ala Thr His Cys Cys Asn AlaAla Arg Thr Gly Gly Tyr Thr Asn 1175 1180 1185 Thr Ala Tyr Gly Ala ArgGly Ala Tyr Ala Thr His Cys Cys Asn 1190 1195 1200 Ala Ala Tyr Ala ThrGly Ala Ala Arg Ala Ala Tyr Trp Ser Asn 1205 1210 1215 Ala Ala Tyr GlyThr Asn Gly Thr Asn Ala Ala Arg Ala Thr Gly 1220 1225 1230 Tyr Thr AsnCys Ala Arg Gly Ala Arg Ala Ala Tyr Trp Ser Asn 1235 1240 1245 Gly AlaArg Tyr Thr Asn Ala Thr Gly Ala Ala Tyr Ala Ala Tyr 1250 1255 1260 AlaAla Tyr Trp Ser Asn Trp Ser Asn Gly Ala Arg Cys Ala Arg 1265 1270 1275Gly Thr Asn Tyr Thr Asn Thr Ala Tyr Gly Thr Asn Gly Ala Tyr 1280 12851290 Cys Cys Asn Ala Thr Gly Ala Thr His Ala Cys Asn Gly Ala Arg 12951300 1305 Ala Thr His Ala Ala Arg Gly Ala Arg Ala Thr His Thr Thr Tyr1310 1315 1320 Ala Thr His Cys Cys Asn Gly Ala Arg Cys Ala Tyr Ala AlaArg 1325 1330 1335 Cys Cys Asn Ala Cys Asn Gly Ala Tyr Thr Ala Tyr AlaAla Arg 1340 1345 1350 Ala Ala Arg Gly Ala Arg Ala Ala Tyr Ala Cys AsnGly Gly Asn 1355 1360 1365 Cys Cys Asn Tyr Thr Asn Gly Ala Arg Ala CysAsn Met Gly Asn 1370 1375 1380 Gly Ala Tyr Thr Ala Tyr Cys Cys Asn CysAla Arg Ala Ala Tyr 1385 1390 1395 Trp Ser Asn Tyr Thr Asn Thr Thr TyrGly Ala Tyr Ala Ala Tyr 1400 1405 1410 Ala Cys Asn Ala Cys Asn Gly ThrAsn Gly Thr Asn Thr Ala Tyr 1415 1420 1425 Ala Thr His Cys Cys Asn GlyAla Tyr Tyr Thr Asn Ala Ala Tyr 1430 1435 1440 Ala Cys Asn Gly Gly AsnThr Ala Tyr Ala Ala Arg Cys Cys Asn 1445 1450 1455 Cys Ala Arg Ala ThrHis Trp Ser Asn Ala Ala Tyr Thr Thr Tyr 1460 1465 1470 Tyr Thr Asn CysCys Asn Gly Ala Arg Gly Gly Asn Trp Ser Asn 1475 1480 1485 Cys Ala TyrTyr Thr Asn Trp Ser Asn Ala Ala Tyr Ala Ala Tyr 1490 1495 1500 Ala AlaTyr Gly Ala Arg Ala Thr His Ala Cys Asn Trp Ser Asn 1505 1510 1515 TyrThr Asn Ala Cys Asn Tyr Thr Asn Ala Ala Arg Cys Cys Asn 1520 1525 1530Cys Cys Asn Gly Thr Asn Gly Ala Tyr Trp Ser Asn Tyr Thr Asn 1535 15401545 Gly Ala Tyr Trp Ser Asn Gly Gly Asn Ala Ala Tyr Ala Ala Tyr 15501555 1560 Cys Cys Asn Met Gly Asn Tyr Thr Asn Cys Ala Arg Ala Ala Arg1565 1570 1575 Cys Ala Tyr Cys Cys Asn Ala Ala Tyr Thr Thr Tyr Gly CysAsn 1580 1585 1590 Thr Thr Tyr Trp Ser Asn Gly Thr Asn Trp Ser Asn TrpSer Asn 1595 1600 1605 Gly Thr Asn Ala Ala Tyr Trp Ser Asn Tyr Thr AsnTrp Ser Asn 1610 1615 1620 Ala Ala Tyr Ala Cys Asn Ala Thr His Thr ThrTyr Tyr Thr Asn 1625 1630 1635 Gly Gly Asn Gly Ala Arg Tyr Thr Asn TrpSer Asn Tyr Thr Asn 1640 1645 1650 Ala Thr His Tyr Thr Asn Ala Ala TyrCys Ala Arg Gly Gly Asn 1655 1660 1665 Gly Ala Arg Thr Gly Tyr Trp SerAsn Trp Ser Asn Cys Cys Asn 1670 1675 1680 Gly Ala Tyr Ala Thr His CysAla Arg Ala Ala Tyr Trp Ser Asn 1685 1690 1695 Gly Thr Asn Gly Ala ArgGly Ala Arg Gly Ala Arg Ala Cys Asn 1700 1705 1710 Ala Cys Asn Ala ThrGly Tyr Thr Asn Tyr Thr Asn Gly Ala Arg 1715 1720 1725 Ala Ala Tyr GlyAla Tyr Trp Ser Asn Cys Cys Asn Trp Ser Asn 1730 1735 1740 Gly Ala ArgAla Cys Asn Ala Thr His Cys Cys Asn Gly Ala Arg 1745 1750 1755 Cys AlaArg Ala Cys Asn Tyr Thr Asn Tyr Thr Asn Cys Cys Asn 1760 1765 1770 GlyAla Tyr Gly Ala Arg Thr Thr Tyr Gly Thr Asn Trp Ser Asn 1775 1780 1785Thr Gly Tyr Tyr Thr Asn Gly Gly Asn Ala Thr His Gly Thr Asn 1790 17951800 Ala Ala Tyr Gly Ala Arg Gly Ala Arg Tyr Thr Asn Cys Cys Asn 18051810 1815 Trp Ser Asn Ala Thr His Ala Ala Tyr Ala Cys Asn Thr Ala Tyr1820 1825 1830 Thr Thr Tyr Cys Cys Asn Cys Ala Arg Ala Ala Tyr Ala ThrHis 1835 1840 1845 Tyr Thr Asn Gly Ala Arg Trp Ser Asn Cys Ala Tyr ThrThr Tyr 1850 1855 1860 Ala Ala Tyr Met Gly Asn Ala Thr His Trp Ser AsnTyr Thr Asn 1865 1870 1875 Tyr Thr Asn Gly Ala Arg Ala Ala Arg 1880 18854 918 PRT Homo sapiens 4 Met Leu Thr Leu Gln Thr Trp Val Val Gln Ala LeuPhe Ile Phe Leu 1 5 10 15 Thr Thr Glu Ser Thr Gly Glu Leu Leu Asp ProCys Gly Tyr Ile Ser 20 25 30 Pro Glu Ser Pro Val Val Gln Leu His Ser AsnPhe Thr Ala Val Cys 35 40 45 Val Leu Lys Glu Lys Cys Met Asp Tyr Phe HisVal Asn Ala Asn Tyr 50 55 60 Ile Val Trp Lys Thr Asn His Phe Thr Ile ProLys Glu Gln Tyr Thr 65 70 75 80 Ile Ile Asn Arg Thr Ala Ser Ser Val ThrPhe Thr Asp Ile Ala Ser 85 90 95 Leu Asn Ile Gln Leu Thr Cys Asn Ile LeuThr Phe Gly Gln Leu Glu 100 105 110 Gln Asn Val Tyr Gly Ile Thr Ile IleSer Gly Leu Pro Pro Glu Lys 115 120 125 Pro Lys Asn Leu Ser Cys Ile ValAsn Glu Gly Lys Lys Met Arg Cys 130 135 140 Glu Trp Asp Gly Gly Arg GluThr His Leu Glu Thr Asn Phe Thr Leu 145 150 155 160 Lys Ser Glu Trp AlaThr His Lys Phe Ala Asp Cys Lys Ala Lys Arg 165 170 175 Asp Thr Pro ThrSer Cys Thr Val Asp Tyr Ser Thr Val Tyr Phe Val 180 185 190 Asn Ile GluVal Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Val Thr 195 200 205 Ser AspHis Ile Asn Phe Asp Pro Val Tyr Lys Val Lys Pro Asn Pro 210 215 220 ProHis Asn Leu Ser Val Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu 225 230 235240 Lys Leu Thr Trp Thr Asn Pro Ser Ile Lys Ser Val Ile Ile Leu Lys 245250 255 Tyr Asn Ile Gln Tyr Arg Thr Lys Asp Ala Ser Thr Trp Ser Gln Ile260 265 270 Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser Ser Phe Thr Val GlnAsp 275 280 285 Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg Ile Arg Cys MetLys Glu 290 295 300 Asp Gly Lys Gly Tyr Trp Ser Asp Trp Ser Glu Glu AlaSer Gly Ile 305 310 315 320 Thr Tyr Glu Asp Arg Pro Ser Lys Ala Pro SerPhe Trp Tyr Lys Ile 325 330 335 Asp Pro Ser His Thr Gln Gly Tyr Arg ThrVal Gln Leu Val Trp Lys 340 345 350 Thr Leu Pro Pro Phe Glu Ala Asn GlyLys Ile Leu Asp Tyr Glu Val 355 360 365 Thr Leu Thr Arg Trp Lys Ser HisLeu Gln Asn Tyr Thr Val Asn Ala 370 375 380 Thr Lys Leu Thr Val Asn LeuThr Asn Asp Arg Tyr Leu Ala Thr Leu 385 390 395 400 Thr Val Arg Asn LeuVal Gly Lys Ser Asp Ala Ala Val Leu Thr Ile 405 410 415 Pro Ala Cys AspPhe Gln Ala Thr His Pro Val Met Asp Leu Lys Ala 420 425 430 Phe Pro LysAsp Asn Met Leu Trp Val Glu Trp Thr Thr Pro Arg Glu 435 440 445 Ser ValLys Lys Tyr Ile Leu Glu Trp Cys Val Leu Ser Asp Lys Ala 450 455 460 ProCys Ile Thr Asp Trp Gln Gln Glu Asp Gly Thr Val His Arg Thr 465 470 475480 Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val 485490 495 Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro Glu Ser Ile Lys Ala500 505 510 Tyr Leu Lys Gln Ala Pro Pro Ser Lys Gly Pro Thr Val Arg ThrLys 515 520 525 Lys Val Gly Lys Asn Glu Ala Val Leu Glu Trp Asp Gln LeuPro Val 530 535 540 Asp Val Gln Asn Gly Phe Ile Arg Asn Tyr Thr Ile PheTyr Arg Thr 545 550 555 560 Ile Ile Gly Asn Glu Thr Ala Val Asn Val AspSer Ser His Thr Glu 565 570 575 Tyr Thr Leu Ser Ser Leu Thr Ser Asp ThrLeu Tyr Met Val Arg Met 580 585 590 Ala Ala Tyr Thr Asp Glu Gly Gly LysAsp Gly Pro Glu Phe Thr Phe 595 600 605 Thr Thr Pro Lys Phe Ala Gln GlyGlu Ile Glu Ala Ile Val Val Pro 610 615 620 Val Cys Leu Ala Phe Leu LeuThr Thr Leu Leu Gly Val Leu Phe Cys 625 630 635 640 Phe Asn Lys Arg AspLeu Ile Lys Lys His Ile Trp Pro Asn Val Pro 645 650 655 Asp Pro Ser LysSer His Ile Ala Gln Trp Ser Pro His Thr Pro Pro 660 665 670 Arg His AsnPhe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe 675 680 685 Thr AspVal Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe 690 695 700 ProGlu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn 705 710 715720 Thr Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser 725730 735 Ser Arg Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn740 745 750 Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly TyrArg 755 760 765 His Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu SerThr Gln 770 775 780 Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu GlnLeu Val Asp 785 790 795 800 His Val Asp Gly Gly Asp Gly Ile Leu Pro ArgGln Gln Tyr Phe Lys 805 810 815 Gln Asn Cys Ser Gln His Glu Ser Ser ProAsp Ile Ser His Phe Glu 820 825 830 Arg Ser Lys Gln Val Ser Ser Val AsnGlu Glu Asp Phe Val Arg Leu 835 840 845 Lys Gln Gln Ile Ser Asp His IleSer Gln Ser Cys Gly Ser Gly Gln 850 855 860 Met Lys Met Phe Gln Glu ValSer Ala Ala Asp Ala Phe Gly Pro Gly 865 870 875 880 Thr Glu Gly Gln ValGlu Arg Phe Glu Thr Val Gly Met Glu Ala Ala 885 890 895 Thr Asp Glu GlyMet Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg Gln 900 905 910 Gly Gly TyrMet Pro Gln 915 5 862 PRT Homo sapiens 5 Met Ala His Thr Phe Arg Gly CysSer Leu Ala Phe Met Phe Ile Ile 1 5 10 15 Thr Trp Leu Leu Ile Lys AlaLys Ile Asp Ala Cys Lys Arg Gly Asp 20 25 30 Val Thr Val Lys Pro Ser HisVal Ile Leu Leu Gly Ser Thr Val Asn 35 40 45 Ile Thr Cys Ser Leu Lys ProArg Gln Gly Cys Phe His Tyr Ser Arg 50 55 60 Arg Asn Lys Leu Ile Leu TyrLys Phe Asp Arg Arg Ile Asn Phe His 65 70 75 80 His Gly His Ser Leu AsnSer Gln Val Thr Gly Leu Pro Leu Gly Thr 85 90 95 Thr Leu Phe Val Cys LysLeu Ala Cys Ile Asn Ser Asp Glu Ile Gln 100 105 110 Ile Cys Gly Ala GluIle Phe Val Gly Val Ala Pro Glu Gln Pro Gln 115 120 125 Asn Leu Ser CysIle Gln Lys Gly Glu Gln Gly Thr Val Ala Cys Thr 130 135 140 Trp Glu ArgGly Arg Asp Thr His Leu Tyr Thr Glu Tyr Thr Leu Gln 145 150 155 160 LeuSer Gly Pro Lys Asn Leu Thr Trp Gln Lys Gln Cys Lys Asp Ile 165 170 175Tyr Cys Asp Tyr Leu Asp Phe Gly Ile Asn Leu Thr Pro Glu Ser Pro 180 185190 Glu Ser Asn Phe Thr Ala Lys Val Thr Ala Val Asn Ser Leu Gly Ser 195200 205 Ser Ser Ser Leu Pro Ser Thr Phe Thr Phe Leu Asp Ile Val Arg Pro210 215 220 Leu Pro Pro Trp Asp Ile Arg Ile Lys Phe Gln Lys Ala Ser ValSer 225 230 235 240 Arg Cys Thr Leu Tyr Trp Arg Asp Glu Gly Leu Val LeuLeu Asn Arg 245 250 255 Leu Arg Tyr Arg Pro Ser Asn Ser Arg Leu Trp AsnMet Val Asn Val 260 265 270 Thr Lys Ala Lys Gly Arg His Asp Leu Leu AspLeu Lys Pro Phe Thr 275 280 285 Glu Tyr Glu Phe Gln Ile Ser Ser Lys LeuHis Leu Tyr Lys Gly Ser 290 295 300 Trp Ser Asp Trp Ser Glu Ser Leu ArgAla Gln Thr Pro Glu Glu Glu 305 310 315 320 Pro Thr Gly Met Leu Asp ValTrp Tyr Met Lys Arg His Ile Asp Tyr 325 330 335 Ser Arg Gln Gln Ile SerLeu Phe Trp Lys Asn Leu Ser Val Ser Glu 340 345 350 Ala Arg Gly Lys IleLeu His Tyr Gln Val Thr Leu Gln Glu Leu Thr 355 360 365 Gly Gly Lys AlaMet Thr Gln Asn Ile Thr Gly His Thr Ser Trp Thr 370 375 380 Thr Val IlePro Arg Thr Gly Asn Trp Ala Val Ala Val Ser Ala Ala 385 390 395 400 AsnSer Lys Gly Ser Ser Leu Pro Thr Arg Ile Asn Ile Met Asn Leu 405 410 415Cys Glu Ala Gly Leu Leu Ala Pro Arg Gln Val Ser Ala Asn Ser Glu 420 425430 Gly Met Asp Asn Ile Leu Val Thr Trp Gln Pro Pro Arg Lys Asp Pro 435440 445 Ser Ala Val Gln Glu Tyr Val Val Glu Trp Arg Glu Leu His Pro Gly450 455 460 Gly Asp Thr Gln Val Pro Leu Asn Trp Leu Arg Ser Arg Pro TyrAsn 465 470 475 480 Val Ser Ala Leu Ile Ser Glu Asn Ile Lys Ser Tyr IleCys Tyr Glu 485 490 495 Ile Arg Val Tyr Ala Leu Ser Gly Asp Gln Gly GlyCys Ser Ser Ile 500 505 510 Leu Gly Asn Ser Lys His Lys Ala Pro Leu SerGly Pro His Ile Asn 515 520 525 Ala Ile Thr Glu Glu Lys Gly Ser Ile LeuIle Ser Trp Asn Ser Ile 530 535 540 Pro Val Gln Glu Gln Met Gly Cys LeuLeu His Tyr Arg Ile Tyr Trp 545 550 555 560 Lys Glu Arg Asp Ser Asn SerGln Pro Gln Leu Cys Glu Ile Pro Tyr 565 570 575 Arg Val Ser Gln Asn SerHis Pro Ile Asn Ser Leu Gln Pro Arg Val 580 585 590 Thr Tyr Val Leu TrpMet Thr Ala Leu Thr Ala Ala Gly Glu Ser Ser 595 600 605 His Gly Asn GluArg Glu Phe Cys Leu Gln Gly Lys Ala Asn Trp Met 610 615 620 Ala Phe ValAla Pro Ser Ile Cys Ile Ala Ile Ile Met Val Gly Ile 625 630 635 640 PheSer Thr His Tyr Phe Gln Gln Lys Val Phe Val Leu Leu Ala Ala 645 650 655Leu Arg Pro Gln Trp Cys Ser Arg Glu Ile Pro Asp Pro Ala Asn Ser 660 665670 Thr Cys Ala Lys Lys Tyr Pro Ile Ala Glu Glu Lys Thr Gln Leu Pro 675680 685 Leu Asp Arg Leu Leu Ile Asp Trp Pro Thr Pro Glu Asp Pro Glu Pro690 695 700 Leu Val Ile Ser Glu Val Leu His Gln Val Thr Pro Val Phe ArgHis 705 710 715 720 Pro Pro Cys Ser Asn Trp Pro Gln Arg Glu Lys Gly IleGln Gly His 725 730 735 Gln Ala Ser Glu Lys Asp Met Met His Ser Ala SerSer Pro Pro Pro 740 745 750 Pro Arg Ala Leu Gln Ala Glu Ser Arg Gln LeuVal Asp Leu Tyr Lys 755 760 765 Val Leu Glu Ser Arg Gly Ser Asp Pro LysPro Glu Asn Pro Ala Cys 770 775 780 Pro Trp Thr Val Leu Pro Ala Gly AspLeu Pro Thr His Asp Gly Tyr 785 790 795 800 Leu Pro Ser Asn Ile Asp AspLeu Pro Ser His Glu Ala Pro Leu Ala 805 810 815 Asp Ser Leu Glu Glu LeuGlu Pro Gln His Ile Ser Leu Ser Val Phe 820 825 830 Pro Ser Ser Ser LeuHis Pro Leu Thr Phe Ser Cys Gly Asp Lys Leu 835 840 845 Thr Leu Asp GlnLeu Lys Met Arg Cys Asp Ser Leu Met Leu 850 855 860

What is claimed is:
 1. A substantially pure or recombinant polypeptidecomprising at least ten contiguous amino acids of the intracellularportion of SEQ ID NO:2.
 2. The polypeptide of claim 1, wherein: a) saidpolypeptide comprises at least 25 contiguous amino acids of theintracellular portion of SEQ ID NO:2; b) said polypeptide isrecombinant, comprising the intracellular portion of SEQ ID NO:2; c)said polypeptide further comprises at least ten contiguous amino acidsof the non-intracellular portion of SEQ ID NO:2; d) said polypeptidecomprises at least 25 amino acids of the extracellular portion of SEQ IDNO:2; e) said polypeptide comprises the mature SEQ ID NO:2; or f) saidpolypeptide is a substantially pure natural polypeptide.
 3. Therecombinant polypeptide of claim 1, which: a) consists of the maturesequence of Table 1; b) is an unglycosylated polypeptide; c) is from ahuman; d) comprises at least 40 contiguous amino acids of SEQ ID NO:2;e) exhibits at least three nonoverlapping segments of at least fifteencontiguous amino acids of SEQ ID NO:2; f) is a natural polymorphicvariant of SEQ ID NO:2; g) has a length at least about 30 amino acids;h) exhibits at least two non-overlapping epitopes which are specific fora primate DCRS5; i) has a molecular weight of at least 30 kD withnatural glycosylation; j) is a synthetic polypeptide; k) is in a sterileform; l) is in an aqueous or buffered solution; m) is attached to asolid substrate; n) is conjugated to another chemical moiety; or o) isphysically associated with an IL-12Rβ1 polypeptide;
 4. A composition ofmatter selected from: a) a substantially pure or recombinant polypeptidecomprising at least two distinct nonoverlapping segments of at least sixcontiguous amino acids of the intracellular portion of SEQ ID NO:2; b) asubstantially pure or recombinant polypeptide comprising at least 12contiguous amino acids of the intracellular poriton of SEQ ID NO:2; orc) a substantially pure natural sequence polypeptide comprising matureSEQ ID NO:2.
 5. The polypeptide: 1) of claim 4a, wherein: a) saiddistinct nonoverlapping segments: i) include one of at least twelveamino acids; ii) include one of at least seven amino acids and a secondof at least nine amino acids; iii) include a third distinct segment ofat least six amino acids; or iv) comprise one of R355-L373, P378-L405,V407-D426, K428-D439, P441-V452, I454-G460, I465-T587, or N592-606; orb) said polypeptide further comprises at least two distinctnonoverlapping segments of at least six contiguous amino acids of theextracellular portion of SEQ ID NO:2; 2) of claim 4b, wherein: a) saidat least twelve contiguous amino acid segment comprises one ofR355-L373, P378-L405, V407-D426, K428-D439, P441-V452, 1454-G460,I465-T587, or N592-606; or b) said polypeptide further comprises atleast two distinct nonoverlapping segments of at least six contiguousamino acids of the extracellular portion of SEQ ID NO:2; or 3) of claim4c, further comprising a purification or detection epitope.
 6. Thepolypeptide of claim 4, which: a) consists of the mature sequence ofTable 1; b) is an unglycosylated polypeptide; c) is from a human; d)comprises at least 40 contiguous amino acids of SEQ ID NO:2; e) exhibitsat least three nonoverlapping segments of at least fifteen contiguousamino acids of SEQ ID NO:2; f) is a natural polymorphic variant of SEQID NO:2; g) has a length at least about 30 amino acids; h) exhibits atleast two non-overlapping epitopes which are specific for a primateDCRS5; i) has a molecular weight of at least 30 kD with naturalglycosylation; j) is a synthetic polypeptide; k) is in a sterile form;l) is in an aqueous or buffered solution; m) is attached to a solidsubstrate; n) is conjugated to another chemical moiety; or o) isphysically associated with an IL-12Rβ1 polypeptide.
 7. A compositioncomprising: a) a substantially pure polypeptide of claim 4 combined withthe IL-12Rβ1 protein; or b) said polypeptide of claim 4 and a carrier,wherein said carrier is: i) an aqueous compound, including water,saline, and/or buffer; and/or ii) formulated for oral, rectal, nasal,topical, or parenteral administration.
 8. A kit comprising a polypeptideof claim 4, and: a) a compartment comprising said polypeptide; b) acompartment comprising an IL-12Rβ1 polypeptide; c) a compartmentcomprising a p40, IL-B30, or p40/IL-B30 polypeptide; or d) instructionsfor use or disposal of reagents in said kit.
 9. A binding compoundcomprising an antigen binding site from an antibody, which specificallybinds to said intracellular portion of said polypeptide of claim 1,wherein: a) said binding compound is in a container; b) said polypeptideis from a human; c) said binding compound is an Fv, Fab, or Fab2fragment; d) said binding compound is conjugated to another chemicalmoiety; or e) said antibody: i) is raised against a peptide sequence ofa mature polypeptide of Table 1; ii) is raised against a mature DCRS5;iii) is raised to a purified human DCRS5; 2 iv) is immunoselected; v) isa polyclonal antibody; luorescent label. vi) binds to a denatured DCRS5;vii) exhibits a Kd to antigen of at least 30 μM; viii) is attached to asolid substrate, including a bead or plastic membrane; ix) is in asterile composition; or x) is detectably labeled, including aradioactive or fluorescent label.
 10. A kit comprising said bindingcompound of claim 9, and: a) a compartment comprising said bindingcompound; b) a compartment comprising: i) a p40 polypeptide; ii) anIL-B30 polypeptide; iii) a DCRS5 polypeptide; and/or iv) an IL-12Rβ1polypeptide; c) a compartment comprising an antibody which bindsselectively to: i) a p40 polypeptide; ii) an IL-B30 polypeptide; iii) aDCRS5 polypeptide; and/or iv) an IL-12Rβ1 polypeptide; or d)instructions for use or disposal of reagents in said kit.
 11. A methodof producing an antigen:antibody complex, comprising contacting underappropriate conditions a primate DCRS5 polypeptide with an antibody ofclaim 9, thereby allowing said complex to form.
 12. The method of claim11, wherein: a) said complex is purified from other cytokine receptors;b) said complex is purified from other antibody; c) said contacting iswith a sample comprising an interferon; d) said contacting allowsquantitative detection of said antigen; e) said contacting is with asample comprising said antibody; or f) said contacting allowsquantitative detection of said antibody.
 13. A composition comprising:a) a sterile binding compound of claim 9, or b) said binding compound ofclaim 9 and a carrier, wherein said carrier is: i) an aqueous compound,including water, saline, and/or buffer; and/or ii) formulated for oral,rectal, nasal, topical, or parenteral administration.
 14. An isolated orrecombinant nucleic acid encoding said DCRS5 polypeptide of claim 1,wherein said: a) DCRS5 is from a human; or b) said nucleic acid: i)encodes an antigenic peptide sequence of Table 1; ii) encodes aplurality of antigenic peptide sequences of Table 1; iii) exhibitsidentity over at least thirteen nucleotides to a natural cDNA encodingsaid segment; iv) is an expression vector; v) further comprises anorigin of replication; vi) is from a natural source; vii) comprises adetectable label; viii) comprises synthetic nucleotide sequence; ix) isless than 6 kb, preferably less than 3 kb; x) is from a primate; xi)comprises a natural full length coding sequence; xii) is a hybridizationprobe for a gene encoding said DCRS5; or xiii) is a PCR primer, PCRproduct, or mutagenesis primer.
 15. A cell or tissue comprising saidrecombinant nucleic acid of claim
 14. 16. The cell of claim 15, whereinsaid cell is: a) a prokaryotic cell; b) a eukaryotic cell; c) abacterial cell; d) a yeast cell; e) an insect cell; f) a mammalian cell;g) a mouse cell; h) a primate cell; or i) a human cell.
 17. A kitcomprising said nucleic acid of claim 14, and: a) a compartmentcomprising said nucleic acid; b) a compartment comprising a nucleic acidencoding: i) a p40 polypeptide; ii) an IL-B30 polypeptide; iii) a DCRS5polypeptide; and/or iv) an IL-12Rβ1 polypeptide; c) a compartmentcomprising: i) a p40 polypeptide; ii) an IL-B30 polypeptide; iii) aDCRS5 polypeptide; and/or iv) an IL-12Rβ1 polypeptide; d) a compartmentcomprising an antibody which selectively binds to: i) a p40 polypeptide;ii) an IL-B30 polypeptide; iii) a DCRS5 polypeptide; and/or iv) anIL-12Rβ1 polypeptide; or e) instructions for use or disposal of reagentsin said kit.
 18. A nucleic acid which: a) hybridizes under washconditions of 30 minutes at 30° C. and less than 2M salt to the portionof SEQ ID NO:1 encoding the intracellular portion; or b) exhibitsidentity over a stretch of at least about 30 nucleotides to theintracellular poriton of a primate DCRS5.
 19. The nucleic acid of claim18, wherein: a) said wash conditions are at 45° C. and/or 500 mM salt;or b) said stretch is at least 55 nucleotides.
 20. The nucleic acid ofclaim 18, wherein: a) said wash conditions are at 55° C. and/or 150 mMsalt; or b) said stretch is at least 75 nucleotides.
 21. A method ofmodulating physiology or development of a cell comprising contactingsaid cell with: a) an antagonist of p40/IL-B30 which is a complexcomprising: i) the extracellular portion of a primate DCRS5; and/or ii)the extracellular portion of a primate IL-12Rβ1; b) an antagonist ofp40/IL-B30 which is an antibody which binds a complex comprising: i)primate DCRS5; and/or ii) primate IL-12Rβ1; c) an antagonist ofp40/IL-B30 which is an antibody which bonds to DCRS5; d) an antagonistof p40/IL-B30 which is an antibody to IL-12Rβ1; e) an antagonist ofp40/IL-B30 which is an antisense nucleic acid to DCRS5 or IL-12Rβ1; orf) an agonist of p40/IL-B30 which is an antibody which binds a complexcomprising: i) primate DCRS5; and/or ii) primate IL-12Rβ1.
 22. Themethod of claim 21, wherein said contacting is with an antagonist, and:a) said contacting is in combination with an antagonist to: i) IL-12;ii) IL-18; iii) TNF; or iv) IFNγ; or b) said cell is from a host which:i) exhibits signs or symptoms of a chronic Th1 mediated disease; ii)exhibits symptoms or signs of multiple sclerosis, rheumatoid arthritis,osteoarthritis, inflammatory bowel disease, diabetes, psoriasis, orsepsis; or iii) receives an allogeneic transplant.
 23. The method ofclaim 21, wherein said contacting is with an agonist, and: a) saidcontacting is in combination with: i) IL-12; ii) IL-18; iii) TNF; or iv)IFNγ; or b) said cell is from a host which: i) exhibits signs orsymptoms of a chronic TH2 response; ii) suffers from a tumor, viral, orfungal growth; iii) receives a vaccine; or iv) suffers from an allergicresponse.